bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unexpected modulation of T cell activity by mechanical cues
Authors: Dada Hannah , Do Kun , Cartagena-Rivera Alexander , Altan-Bonnet Grégoire 
Published: November 2024

Abstract
Current immunotherapies take advantage of the biochemical features of T cell activation and function, such as strengthening the antigenic responses of T cells. However, the biophysical properties and the physical environment of T cells are also relevant. Using polyacrylamide (PAA) gels to vary the mechanical landscape, we show that the mechanical environment of T cells dictates T cell capacity. Mechanical properties of PAA gels include viscoelasticity, porosity, and stiffness. Using a robotic platform that enables the automatic collection of cytokines from T cells co-cultured with tumors of varying antigenic strengths, we show that not only does antigenic quality influence T cell function, but altering the crosslinking of the PAA gel, and thus the physical environment, results in varied T cell response. Low crosslinking impairs T cell function, while increasing crosslinking gradually improves response. By using other hydrogels, we ruled out stiffness as a mediator of T cell response. Rheology and atomic force microscopy (AFM) will help uncover the physical characteristics that dictate response. Modeling these properties is essential in identifying the mechanical landscape responsible for immune response and predicting this response based on the tumor physical environment. These findings will introduce a novel approach to treating cancer by assessing the mechanical properties of the tumor and changing the physical environment to one that allows for a favorable immune response.
SLAMF7+ CD8+ T cells exhibit decreased anti-tumor responses in renal cell carcinoma
Authors: Wirth Lena , Hugaboom Miya , Street Kelly , Ruthen Neil , Jegede Opeyemi , Schindler Nicholas , McDermott David , Plimack Elizabeth , Sosman Jeffrey , Haas Naomi , Hurwitz Michael , Hammers Hans , Signoretti Sabina , Atkins Michael , Wu Catherine , Braun David 
Published: November 2024

Abstract
Despite the success of immune-checkpoint inhibitors (ICI) for the treatment of renal cell carcinoma (RCC), many patients fail to receive durable clinical benefits. Therefore, an understanding of resistance to ICIs in RCC is critical for the treatment of this disease. Through single-cell transcriptomic analysis of patient T cells in the HCRN GU16-260 trial, we previously identified a population of CD8+ T cells expressing the SLAMF7 immune receptor that was associated with resistance to nivolumab monotherapy. Here, we functionally interrogated the impact of SLAMF7 overexpression on CD8+ T cell effector functions and found a reduction of pro-inflammatory cytokine and Granzyme B (GzmB) production. CD3+ T cells from healthy human PBMCs were isolated, transduced with either a SLAMF7-GFP or control GFP lentiviral construct, sorted for GFP expression, and subsequently incubated with a SLAMF7 ligand or control. Cytokine and GzmB production were measured using intracellular staining by flow cytometry. SLAMF7-GFP+ CD8+ T cells incubated with a SLAMF7 ligand for 8 hours exhibited a 41.1% reduction in IFN-γ (p=0.0129), 38.3% reduction in IL-2 (p=0.0483), and a 43.8% reduction in GzmB (p=0.0044) production compared to control GFP+ CD8+ T cells. Future work will interrogate the impact of SLAMF7 expression on CD8+ T cell effector functions using patient RCC tumor-infiltrating lymphocytes and evaluate the efficacy of a therapeutic SLAMF7 blocking antibody in restoring anti-tumor functions.
Spatio-temporal Phenotyping of Murine Embryos with Ultrahigh-plex Antibody Panels for Characterization of Early Development
Authors: Tam Tsz , Cheikh Bassem , Nikulina Nadezhda , Klymyshyn Dmytro , Ma Ning , Jhaveri Niyati 
Published: November 2024

Abstract
The murine embryo model is one of the most common animal models for studying developmental biology. Embryonic development is a highly regulated process with spatial and temporal expression of proteins for regional specialization and tissue-specific functions. Application of spatial proteomics to developing embryos enables us to understand cellular organization and interactions within tissues along with regulatory mechanisms at the single-cell level. In this study, we applied a 36-plex panel, specifically designed for analysis of mouse embryos at different stages of development using the PhenoCycler™-Fusion (PCF) 2.0 Platform. This panel was developed by selecting biomarkers for immune cell lineages, immune activation, proliferation, structural and neuronal components. Purified antibodies were tagged to PhenoCycler barcodes and verified for specificity and reproducibility. After successful validation, the panel was deployed on the FFPE mouse embryos and ultrahigh-plex whole-slide imaging was performed using the PCF 2.0 platform. Unsupervised clustering analysis and deep phenotyping were performed to generate a high-resolution spatial atlas of mouse embryos at different stages of development. The tools developed as part of this study can be used to conduct in-depth research on various aspects of organogenesis and to study the spatio-temporal dynamics of cellular and molecular expression in developing embryos.
Alternative cell counting and viability detection using NexGreen/PI fluorescent stain on multiple low- and high-throughput image cytometry platforms
Authors: Nitta Carolina , Pierce Mackenzie , Hem Sopaul , Browe Daniel , Kuksin Dmitry , Lin Bo 
Published: November 2024

Abstract
In the past decade, the increase in cell and gene therapy products such as biologics, antibodies, and CAR-T has significantly increased the need for precise, robust, and consistent cell counting and viability measurements. In this work, we demonstrate a novel fluorescence-based dye, NexGreen/PI, capable of live and dead cell detection comparable to AO/PI in multiple cell buffer/media conditions. Jurkat cells of different viabilities (low, intermediate, and high) were stained in parallel with either NexGreen/PI or AO/PI for 1-2 minutes and measured on multiple image cytometer platforms including low-throughput and high-throughput instruments. Both NexGreen/PI and AO/PI dyes are used as equal volumes added to cell samples. Additionally, we tested if difficult to analyze cells in “spent” media (typically with a lower pH) or in PBS can be stained using NexGreen/PI compared to AO/PI. Results on the high-throughput platforms show that NexGreen/PI staining can discriminate between live and dead Jurkats, analogous to AO/PI. Measured viability and cell concentration of all samples using NexGreen/PI was within 5% of the paired AO/PI-stained samples. Low-throughput instrument results are also similar. Our experiments demonstrate that NexGreen/PI is scalable and can be of significant value for use in automation and high-throughput systems where multiple samples in different mediums can be precisely measured.
Generation of targeted antibody conjugates using Sortase and the AbFlex technology platform.
Authors: Jelinek Mary Anne , Tchoub Mike , Park Hyun Min , Truong Vi , Egan Brian , Weber Julie , Huang Chengyang , Bergseid Jacqueline 
Published: November 2024

Abstract
The miniaturization of experiments to the single cell scale and a growing interest in multiplexing analyte detection is a major driver for the development of novel antibody conjugation technologies that are robust, and which also preserve antibody function. Relative to two step detection modalities, direct primary antibody conjugates provide enhanced specificity and selectivity. We have developed the AbFlex platform, a versatile epitope tagging system for the carboxyl terminus of recombinant mouse and rabbit immunoglobulin heavy chains. It consists of a tandem array of three tags: 1) The Sortase tag recognition sequence (LPXTG); 2) The motif recognized by the BirA biotin ligase, followed by 3) the 6XHis Tag, which can be used with nickel-based purification systems.
Sortase A belongs to a family of transpeptidases found in Gram-positive bacteria that catalyze the attachment of poly-Glycine labels to the Sortase recognition sequence under physiological conditions and with reaction times of approximately one hour. We describe the use of the Staphylococcus aureus Sortase A5 pentamutant that is significantly more active than wild-type Sortase to provide a faster, more efficient labeling reaction. Performance of various recombinant mouse and rabbit antibody conjugates in a variety of applications will be presented.
Characterizing the health of transduced CAR-T cells using high-throughput image cytometry
Authors: Huang Yongyang , Guo Ning , Patel Samir , Pierce Mackenzie , Chan Leo , Lin Bo , Yang Yi 
Published: November 2024

Abstract
Chimeric antigen receptor (CAR)-T cell therapy is one of the novel cellular therapeutic options for cancer patients. In this work, we developed a rapid cell viability and apoptosis detection method to characterize the cell health and recovery of SubT1 cells after the plasmid-based electroporation process to create CAR expression cells using the Cellaca™ PLX Image Cytometer. Viability measurement of transduced SupT1 cells was performed by staining with acridine orange/propidium iodide (AO/PI). In addition, early and late-stage apoptosis were detected by staining transduced SupT1 cells with Annexin V/PI and Caspase3/RubyDead, respectively. Results showed that the introduction of plasmids, other than the electroporation process itself, may induce 20 – 30% additional cell death in the process, indicated by viability results measured on Day 1 post transduction. Among all the Annexin V or Caspase3 positive transduced SupT1 cells on Day 1 (22 – 34%), 70 - 80% of these cells were double positive in both Annexin V and PI, or both Caspase3 and RubyDead, and only 20 – 30% of them were in the early-stage/late-stage apoptosis. Subsequently, the SupT1 cells were quickly recovered with their viabilities increased to &gt;90% on Day 3. With such capability of rapid detection of cell viability and apoptosis, Cellaca™ PLX Image Cytometer can potentially support the characterization of factors that affects the quality of CAR-T cells in the manufacturing process.
LAG3 regulates antibody responses following kidney transplantation
Authors: Nicosia Michael , Fan Ran , Lee Juyeun , Gorbacheva Victoria , Valenzuela Jose , Yamamoto Yosuke , Beavers Ashley , Dvorina Nina , Chuluyan Eduardo , Araki Motoo , Baldwin III William , Fairchild Robert , Min Booki , Valujskikh Anna 
Published: November 2024

Abstract
Lymphocyte activation gene-3 (LAG3) is a coinhibitory receptor expressed by a range of immune cells. The immunomodulatory potential of LAG3 is being explored in cancer and autoimmunity fields but not in transplantation. Using our mouse kidney transplant models we sought to address the role of LAG3 in graft rejection.
Untransplanted LAG3-/- mice have elevated heterologous immunity against a panel of alloantigens. Recipient LAG3 deficiency results in rapid rejection of MHC-mismatched renal allografts that are spontaneously accepted by WT recipients, with graft histology characteristic of antibody mediated rejection (ABMR). Antagonistic LAG3 antibody treatment of WT recipients enhanced anti-donor immune responses and induced kidney damage typical of chronic rejection, indicating that LAG3 regulates de novo alloresponses. Recipient B cell depletion, but not CD8+ T cells, prevented kidney allograft rejection in LAG3-/- recipients further supporting ABMR as the mechanism of graft loss. Posttransplant, follicular T cells and plasma cells are among LAG3 expressing immune cells. To identify the mechanism we used T or B cell conditional LAG3 knockout recipients, neither reject the allograft but both had elevated levels of donor specific antibodies (DSA), indicating LAG3 expression on either T or B cells is sufficient to regulate anti-donor humoral immunity. Our results identify LAG3 as a regulator of DSA following kidney transplantation and a potential therapeutic target for ABMR.
Reversing pregnancy induced humoral sensitization to facilitate transplantation tolerance
Authors: Durgam Samarth , Wang Qiang , Yin Dengping , Alegre Maria Luisa , Chong Anita 
Published: November 2024

Abstract
Purpose: Pregnancy induces split tolerance with tolerization of fetus-specific effector T cells, but sensitization of B cells contributing to sex disparity in access to transplantation for multiparous women. We previously showed that, following mating with Balb/c males, post-partum sIgKO mice (mIgM-/-AID-/-) that cannot produce secreted antibodies were tolerized by aCD154 and accepted F1 heart grafts (TolRx); this was not achieved with WT mice. Here, we tested, 1) if B cells from these sIgKO mice acquired cell intrinsic hypofunction? 2) if post-partum serum inhibited the acquisition and reversed intrinsic hypofunction in post-partum B cells? Methods: Post-partum sIgKO mice were subjected to TolRx ± transfused post-partum serum. After 30 days, B cells were transferred to MD4 hosts and challenged with F1 splenocytes ± post-partum serum. F1-specific germinal center B cells were quantified on D-14. Results: 1) Post-partum B cells subject to TolRx did not differentiate into germinal center B cells in MD4 mice, in contrast to non -tolerized post-partum B cells that did. 2) Post-partum serum transfused during TolRx or into MD4 mice inhibited the induction and reversed hypofunction in post-partum B cells. Conclusion: Pregnancy-sensitized B cells develop a state of intrinsic hypofunction when tolerance was induced with aCD154 to F1 HTx but was antagonized by fetus-specific antibodies.
Toddlers have limited mucosal protection against Live Attenuated Influenza Virus in human tonsil organoids.
Authors: Sun Meng , Sola Elsa , Guo Jing , Shankar Vishnu , Kamalyan Lilit , Gupta Neha , Capasso Robson , Davis Mark 
Published: November 2024

Abstract
Children require separate clinical trials to vaccination and other treatments, because we know little about how their immune systems differ from adults. Notably, we developed a high-resolution in-vitro model of human immunity using tonsil organoids. These organoids have demonstrated a remarkable ability to generate specific antibody and T-cell responses to vaccines across a broad age spectrum (2-70 years old). This provides a unique opportunity to investigate the variations to vaccination between children and adults. To study the systemic cellular response to live attenuated influenza virus in human tonsil organoids, we used scRNA-seq via the BD Rhapsody platform, and validated by flow cytometry, ELISA and Luminex assays. Our data showed marked differences between toddlers (2-4 years) and adults (19-39 years). Compared to adults, toddlers exhibited lower levels of flu-specific IgA, IgG antibodies and activated cytotoxic CD8 T cells. Additionally, toddlers displayed a less comprehensive cytokines that help promote class-switched antibody production. Our data also indicated a metabolic transition within germinal centers from oxidative phosphorylation to glycolysis, as individuals progress from early childhood to adulthood. Our study can offer valuable insights to aid vaccine design and administration practices. It may also identify vulnerabilities in children's immune systems with implications for improving therapeutic treatments for infectious diseases.
Monoclonal antibody-mediated control of lyssavirus infection in the CNS requires Fc effector function
Authors: Huaman Celeste , Mastraccio Kate , Clouse Caitlyn , Rader Maddie , Swafford Isabella , Yan Lianying , Smith Ina , Markotter Wanda , Broder Christopher , Schaefer Brian 
Published: November 2024

Abstract
Lyssaviruses are the causative agent for rabies, a disease that is uniformly fatal in humans. Once infection reaches the central nervous system (CNS), currently approved therapies are ineffective. Using luminescence-based longitudinal tracing of lyssavirus infection in a mouse model of lethal rabies disease, we have shown that peripheral administration of a single dose of human monoclonal antibody (mAb) F11 protects animals from lethality, even when virus is already robustly replicating in the CNS. Additionally, behavioral analyses demonstrate improved motor function of the infected mAb-treated animals compared to infected, untreated controls. Interestingly, A6, another huIgG1 mAb that targets the same lyssavirus G epitope and exhibits similar in vitro neutralization efficiency, is inferior to F11 when used for in vivo therapy. Importantly, functional inactivation of the F11 Fc domain significantly impairs protection against mortality. Thus, Fc effector function is a major determinant of mAb in vivo efficacy. Histology and flow cytometry analysis shows that mAb therapy leads to a shift in leukocyte populations that infiltrate the brain, with significant increases in MHCII+ myeloid cells and CD4+ T cells, consistent with our demonstration that CD4+ T cells are essential for mAb efficacy. Collectively, these data suggest that Fc-mediated effector functions in the periphery yield a potent antiviral immune response in the CNS.
Fractionation of mRNA vaccines improves immune responses
Authors: Sanchez Sarah , Awakoaiye Bakare , Dangi Tanushree , Irani Nahid , Penaloza-MacMaster Pablo 
Published: November 2024

Abstract
mRNA vaccines are used to prevent SARS-CoV-2 infection and are being explored for other infectious diseases as well as cancer. While they have shown high efficacy at preventing COVID-19, waning immunity has motivated the development of improved mRNA vaccine formulations. Vaccine fractionation has been explored previously in the context of viral vector and protein immunogens, but its effect on mRNA vaccines is still unclear. We interrogated whether fractionating an mRNA-SARS-CoV-2 spike vaccine over three days, as opposed to a single dose, would enhance immune responses in C57BL/6 mice. We compared the immune responses induced by three consecutive 1 μg doses over three days (fractionated vaccine) with a single 3 μg dose. Despite the equivalent overall dosage, the fractionated vaccine demonstrated superior CD8 T cell responses. This pattern of enhanced immune responses with the fractionated vaccine regimen was also observed with an mRNA-HIV vaccine, suggesting generalizability. Taken together, these findings suggest that prolonging the duration of antigen expression through mRNA vaccination could enhance immune responses, supporting the rationale for developing slow-release formulations for mRNA vaccines.
Sex differences in protection conferred by heterologous vaccines for pneumonic plague in an aerosol challenge model
Authors: Davies Michael , Biryukov Sergei , Rill Nathaniel , Klimko Christopher , Dankmeyer Jennifer , Hunter Melissa , Miller Jeremy , Shoe Jennifer , Talyansky Yuli , Qiu Ju , Bozue Joel , Cote Christopher 
Published: November 2024

Abstract
Pneumonic plague, caused by the bacterium Yersinia pestis, is a severe and rapidly progressing illness transmitted via aerosol. There are no FDA-approved vaccines, and candidate vaccines may be less effective against pneumonic than bubonic plague. Y. pestis is not known to impact males and females differently in mechanism of pathogenesis or severity. However, a recent study reported sex-biased vaccine effectiveness after intranasal challenge. In this study we observed that when male mice were given a vaccine that is protective in females challenged with aerosolized virulent Y. pestis C12, males were not protected. We then gave male and female BALB/c mice a recently developed heterologous vaccine strategy and monitored survival, bacterial burden (CFU), and immunological factors before and after aerosol challenge. This strategy consisted of two subcutaneous injections: one of Y. pestis live attenuated vaccine (LAV) strain pgm-pPst-Δcaf1 or pgm-pPst-ΔyopD/Δcaf1 and one of rF1-V protein subunit with adjuvants CpG and Alhydrogel. When primed with rF1-V followed by pgm-pPst-Δcaf1 LAV boost, 100% of females and 50% of males survived challenge. This sex difference was observed to a lesser extent with three other vaccines. Males had higher CFU titers and differing patterns of serum antibodies, lung cytokines, and splenic T cells. These data provide new knowledge about sex differences in vaccine efficacy and immunological factors that differ between males and females.
A systems vaccinology resource to develop and test computational models of immunity
Authors: Shinde Pramod , Willemsen Lisa , Orfield Shelby , Kojima Mari , Reyna Joaquin , Ha Brendan , Greenbaum Jason , Overton James , Nili Somayeh , Rasmussen Mikkel , Gygi Jeremy , Antunes Ricardo da Silva , Sette Alessandro , Olsen Lars Ronn , Grant Barry , Konstorum Anna , Guan Leying 
Published: November 2024

Abstract
Computational models that predict an individual's response to a vaccine offer the potential for mechanistic insights and personalized vaccination strategies. These models often stem from small cohort studies focusing on single vaccines limiting their generalizability. The ability to assess the performance of resulting models would be improved by comparing their performance on independent datasets. We established a prototype platform that evaluates Computational Models of Immunity to Pertussis Booster vaccinations (CMI-PB). It aims to generate experimental data specifically for model assessment through annual data releases and contests. In a preliminary 'dry run', over 30 existing computational models were tested to predict immune responses from pre-vaccination multi-omic profiles with only one successful model based on age. The performance of new models built using CMI-PB training data was much better but varied significantly based on the choice of pre-vaccination features used and the model-building strategy. This suggests that previously published models developed for other vaccines do not generalize well to Pertussis Booster vaccination. Overall, these results reinforced the need for comparative analysis across models and datasets, which CMI-PB aims to achieve. We seek wider community engagement for our first public prediction contest, which will open in mid 2024.
Describing Zoom exhaustion and fatigue in nursing students at a university in South Africa
Authors: Emmamally Waheedha , Wentzel Dorien , Brysiewicz Petra 
Published: November 2024

Background: Online platforms have gained considerable support from students and lecturers post COVID-19, however they are associated with student exhaustion and fatigue.Aim: To determine Zoom exhaustion and fatigue in nursing students at a selected university in South Africa.Methods: A quantitative cross - sectional study was conducted in KwaZulu-Natal to collect data from 146 nursing students registered at the selected university for the 4-year Bachelor of Nursing programme. The Zoom Exhaustion and Fatigue Scale collected data on responses to domains of general fatigue, visual fatigue, social fatigue, motivational fatigue, and emotional fatigue. The data were analysed using the International Business Machines, Statistical Package for Social Sciences, version 26.0. Descriptive statistics were calculated for demographics, a total score was calculated and mean scores and 95% confidence intervals for the different domains were calculated. Mann Whitney U and Kruskal Wallis Independent Tests were calculated to determine associations between demographics and Zoom usage.Results: The overall Zoom exhaustion and fatigue score was 46.71 (s.d. = 10.50). The motivational fatigue construct had the highest mean score of 3.29 (s.d. = 0.83), followed by general (3.18 s.d. = 0.81), social (3.11, s.d. = 0.88), emotional (3.06, s.d. = 0.99) and visual fatigue (2.92, s.d. = 0.94).Conclusion: The study concluded that while the overall mean score indicated moderate levels of Zoom exhaustion and fatigue among respondents, majority of the respondents scored high levels ( 48) of Zoom fatigue. Students reported higher levels of motivational fatigue compared to the other 4 constructs.