CellCelector™ as a platform in isolating primary B cells for antibody discovery

Background The most robust strategy in antibody discovery is the use of immunized animals and the ability to isolate and immortalize immune B-cells to hybridoma for further interrogation. However, capturing the full repertoire of an immunized animal is labor intensive, time consuming, and limited in throughput. Therefore, techniques to directly mine the antibody repertoire of primary B-cells are of great importance in antibody discovery. Methods In the current study, we present a method to isolate individual antigen specific primary B-cells using the CellCellector™ single-cell isolation platform from XenoMouse® (XM) immunized with a recombinant therapeutic protein, EGFR. We screened a subset of CD138+ B-cells and identified 238 potential EGFR specific B-cells from 1,189 antibody secreting cells (ASCs) and isolated 94 by CellCellector. Results We identified a diverse set of heavy chain CDR sequences and cloned and expressed 20 into a standard human IgG1 antibody format. We further characterized and identified 13 recombinant antibodies that engage soluble and native forms of EGFR. By extrapolating the method to all 400,000 CD138+ B-cells extracted from one EGFR immunized XM, a potential 1,196 unique EGFR-specific antibodies could be discovered. Conclusions CellCelector allows for interrogating the B-cell pool directly and isolating B-cells specific to the therapeutic target of interest. Furthermore, antibody sequences recovered from isolated B-cells engage the native and recombinant target, demonstrating the CellCellector can serve as a platform in antibody discovery.

Satellite Cells Exhibit Decreased Numbers and Impaired Functions on Single Myofibers Isolated from Vitamin B6-Deficient Mice

Emerging research in human studies suggests an association among vitamin B6, sarcopenia, and muscle strength. However, very little is known regarding its potential role at the cellular level, especially in muscle satellite cells. Therefore, to determine whether vitamin B6 affects the satellite cells, we isolated single myofibers from muscles of vitamin B6-deficient and vitamin B6-supplemented mice. Subsequently, we subjected them to single myofiber culture and observed the number and function of the satellite cells, which remained in their niche on the myofibers. Prior to culture, the vitamin B6-deficient myofibers exhibited a significantly lower number of quiescent satellite cells, as compared to that in the vitamin B6-supplemented myofibers, thereby suggesting that vitamin B6 deficiency induces a decline in the quiescent satellite cell pool in mouse muscles. After 48 and 72 h of culture, the number of proliferating satellite cells per cluster was similar between the vitamin B6-deficient and -supplemented myofibers, but their numbers decreased significantly after culturing the myofibers in vitamin B6-free medium. After 72 h of culture, the number of self-renewing satellite cells per cluster was significantly lower in the vitamin B6-deficient myofibers, and the vitamin B6-free medium further decreased this number. In conclusion, vitamin B6 deficiency appears to reduce the number of quiescent satellite cells and suppress the proliferation and self-renewal of satellite cells during myogenesis.

Quantification of T-cell dynamics during latent cytomegalovirus infection in humans

Cytomegalovirus (CMV) infection has a major impact on the T-cell pool, which is thought to be associated with ageing of the immune system. The effect on the T-cell pool has been interpreted as an effect of CMV on non-CMV specific T-cells. However, it remains unclear whether the effect of CMV could simply be explained by the presence of large, immunodominant, CMV-specific memory CD8+ T-cell populations. These have been suggested to establish through gradual accumulation of long-lived cells. However, little is known about their maintenance. We investigated the effect of CMV infection on T-cell dynamics in healthy older adults, and aimed to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8+ T-cells. We studied the expression of senescence, proliferation, and apoptosis markers and quantified the in vivo dynamics of CMV-specific and other memory T-cell populations using in vivo deuterium labelling. Increased expression of late-stage differentiation markers by CD8+ T-cells of CMV+ versus CMV- individuals was not solely explained by the presence of large, immunodominant CMV-specific CD8+ T-cell populations. The lifespans of circulating CMV-specific CD8+ T-cells did not differ significantly from those of bulk memory CD8+ T-cells, and the lifespans of bulk memory CD8+ T-cells did not differ significantly between CMV- and CMV+ individuals. Memory CD4+ T-cells of CMV+ individuals showed increased expression of late-stage differentiation markers and decreased Ki-67 expression. Overall, the expression of senescence markers on T-cell populations correlated positively with their expected in vivo lifespan. Together, this work suggests that i) large, immunodominant CMV-specific CD8+ T-cell populations do not explain the phenotypical differences between CMV+ and CMV- individuals, ii) CMV infection hardly affects the dynamics of the T-cell pool, and iii) large numbers of CMV-specific CD8+ T-cells are not due to longer lifespans of these cells.

Failures in thymus medulla regeneration during immune recovery cause tolerance loss and prime recipients for auto-GVHD

Bone marrow transplantation (BMT) is a widely used therapy for blood cancers and primary immunodeficiency. Following transplant, the thymus plays a key role in immune reconstitution by generating a naive αβT cell pool from transplant-derived progenitors. While donor-derived thymopoiesis during the early post-transplant period is well studied, the ability of the thymus to synchronize T cell development with essential tolerance mechanisms is poorly understood. Using a syngeneic mouse transplant model, we analyzed T cell recovery alongside the regeneration and function of intrathymic microenvironments. We report a specific and prolonged failure in the post-transplant recovery of medullary thymic epithelial cells (mTECs). This manifests as loss of medulla-dependent tolerance mechanisms, including failures in Foxp3+ regulatory T cell development and formation of the intrathymic dendritic cell pool. In addition, defective negative selection enables escape of self-reactive conventional αβT cells that promote autoimmunity. Collectively, we show that post-transplant T cell recovery involves an uncoupling of thymopoiesis from thymic tolerance, which results in autoimmune reconstitution caused by failures in thymic medulla regeneration.

SORLA is required for insulin-induced expansion of the adipocyte precursor pool in visceral fat

Visceral adipose tissue shows remarkable plasticity, constantly replacing mature adipocytes from an inherent pool of adipocyte precursors. The number of precursors is set in the juvenile organism and remains constant in adult life. Which signals drive precursor pool expansion in juveniles and why they operate in visceral but not in subcutaneous white adipose tissue (WAT) are unclear. Using mouse models, we identified the insulin-sensitizing receptor SORLA as a molecular factor explaining the distinct proliferative capacity of visceral WAT. High levels of SORLA activity in precursors of juvenile visceral WAT prime these cells for nutritional stimuli provided through insulin, promoting mitotic expansion of the visceral precursor cell pool in overfed juvenile mice. SORLA activity is low in subcutaneous precursors, blunting their response to insulin and preventing diet-induced proliferation of this cell type. Our findings provide a molecular explanation for the unique proliferative properties of juvenile visceral WAT, and for the genetic association of SORLA with visceral obesity in humans.

Loss of Atg2b and Gskip impairs the maintenance of the hematopoietic stem cell pool size

A germline copy number duplication of chromosome 14q32, which contains ATG2B and GSKIP , was identified in families with myeloproliferative neoplasm (MPN). Herein, we show that mice lacking both Atg2b and Gskip , but not either alone, exhibited decreased hematopoiesis, resulting in death in utero accompanied by anemia. In marked contrast to MPN patients with duplication of ATG2B and GSKIP , the number of hematopoietic stem cells (HSCs), in particular long-term HSCs, in double knockout fetal livers were significantly decreased due to increased cell death. Although the remaining HSCs still had the ability to differentiate into hematopoietic progenitor cells, the differentiation efficiency was quite low. Remarkably, mice with knockout of Atg2b or Gskip alone did not show any hematopoietic abnormality. Mechanistically, while loss of both genes had no effect on autophagy, it increased the expression of genes encoding enzymes involved in oxidative phosphorylation. Taken together, our results indicate that Atg2b and Gskip play a synergistic effect in maintaining the pool size of HSCs.

A Multicompartment Mathematical Model Based on Host Immunity for Dissecting COVID-19 Heterogeneity

Background: As of early August 2021, more than 190 million people have developed coronavirus disease (COVID-19), a pandemic that has killed approximately 4 million people. Caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 exhibited a highly variable clinical course, ranging from a high proportion of asymptomatic and mild infections to severe and fatal disease. However, the immunological determinants underlying the heterogeneity of COVID-19 remain to be fully elucidated. Methods: To systemically analyze the immunopathogenesis of COVID-19, a multicompartment mathematical model based on both immunological principles and COVID-19-related work performed by the scientific community was built to illustrate the dynamics of host immunity after SARS-CoV-2 infection. We used ordinary differential equations (ODEs) to simulate the time-dependent functions of immunologic variations in the four compartments, which were draining lymph nodes, peripheral blood, lung and distant lymph nodes and spleen. Our model consisted of equations for 109 immunologic variations, which contained 223 parameters. K was used to characterize the adequacy of the SARS-CoV-2-specific naïve T/B cell pool; K I represented the hill coefficient of antigen-presenting cell (APC) differentiation. Further, we used method of pseudo landscape to visualize the effect of APC capacity and the SARS-CoV-2-specific naïve T/B cell pool on clinical outcomes. Results: Based on both immunologic knowledge and extensive COVID-19-related work performed by the scientific community, we constructed a knowledge-driven mathematical model that incorporated SARS-CoV-2 infection, bacterial infection, leukocyte chemotaxis, innate immunity and adaptive immunity. The model simulated and predicted the different trajectories of the viral load, bacterial load, immune cells, cytokines and infected epithelial cells in patients with different severities. A higher viral load and longer virus-shedding period were observed in patients with higher severity, along with an increase in SARS-CoV-2-infected lung epithelial cells. The trajectories of both peripheral blood IL-6 and lymphocytes predicted COVID-19 outcomes. Based on the distribution, trafficking and differentiation of immune cells after SARS-CoV-2 infection, we proposed that early-stage lymphopenia is related to lymphocyte chemotaxis. The delayed initiation of both innate and adaptive immunity resulted in elevated SARS-CoV-2 shedding and was a pivotal cause of COVID-19 severity. Spatiotemporally, viral shedding and postviral bacterial infection evoked stronger innate immunity. Viral shedding could be restrained by the rapid initiation of APC, antibody-secreting cell (ASC) and cytotoxic T cell (CTL). Moreover, our model predicted that the insufficient SARS-CoV-2-specific naïve T/B cell pools and inactive APC caused a series of chain reactions, including viral shedding, bacterial infection, sepsis and cytokine storms. Finally, pseudopotential analysis revealed that a high state characterized by severe bacterial infections and cytokine storms was a stable attractor for patients with insufficient SARS-CoV-2-specific naïve T/B cells and inactive APC (Figure 1). Conclusion: Overall, our analysis provided a comprehensive view of the dynamics of host immunity after SARS-CoV-2 infection and highlighted that the antigen-specific naïve T/B cell pool and APC ability may essentially determine COVID-19 heterogeneity from an immunological standpoint. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Planarians as an In Vivo Experimental Model for the Study of New Radioprotective Substances

Ionising radiation causes the death of the most actively dividing cells, thus leading to depletion of the stem cell pool. Planarians are invertebrate flatworms that are unique in that their stem cells, called neoblasts, constantly replace old, damaged, or dying cells. Amenability to efficient RNAi treatments, the rapid development of clear phenotypes, and sensitivity to ionising radiation, combined with new genomic technologies, make planarians an outstanding tool for the discovery of potential radioprotective agents. In this work, using the well-known antioxidant N-acetylcysteine, planarians are, for the first time, shown to be an excellent model system for the fast and effective screening of novel radioprotective and radio-sensitising substances. In addition, a panel of measurable parameters that can be used for the study of radioprotective effects on this model is suggested.

SOLAR POWER PLANT PLANNING WITH TILE MODIFICATION FOR SOLAR PANEL INSTALLATION

Solar power plants have been created using solar cells as power plants. This power plant utilizes the source of sunlight as its source.solar cell as receiving sunlight as a source of electricity. Utilization of sunlight to become electrical energy, Designed from tile as a medium and solar cell as a receiver of solar energy into electrical energy. Where batrai as a charging to be used, this tile as a tool planted solar cell so that it can be used tools that generate electrical energy. Solar power is one of the environmentally friendly renewable energy sources. Solar power is utilized by solar power plants to generate electricity. The electrical energy generated is the light energy converted by solar cells. The solar cell pool is arranged in such a way that it produces solar panels. The resulting electrical energy will be stored in a medium called.

Visceral mesoderm signaling regulates assembly position and function of the Drosophila testis niche

SummaryTissue homeostasis often requires a properly placed niche to support stem cells. The morphogenetic processes that position a niche are just being described. We recently showed that Drosophila testis pro niche cells, specified at disparate positions during early gonadogenesis, must assemble in one collective at the gonad anterior. Here, we identify Slit and FGF signals emanating from adjacent visceral mesoderm (Vm) that regulate assembly. In response to signaling, niche cells express islet, which we find is also required for positioning the niche. Without signaling, niche cells specified furthest from the anterior are unable to migrate, remaining dispersed. Function of the dispersed niche is severely disrupted, with pro-niche cells evading cell cycle quiescence, compromised in their ability to signal the incipient stem cell pool, and failing to orient stem cell divisions properly. Our work identifies both extrinsic signaling and intrinsic responses required for proper assembly and placement of the testis niche.