bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeted transcriptome sequencing enables exponential scaling of combinatorial barcoding in leukemia samples
Authors: Kim Grace , Papalexi Efthymia , Matulich Peter , Koehler Ryan , Schroeder Sarah , Tran Vuong , Roco Charles , Rosenberg Alexander 
Published: August 2023

Abstract
Single-cell RNA-sequencing (scRNA-seq) has rapidly spread across multiple research fields, leading to new discoveries. Many applications of scRNA-seq are focused on cell type identification, gene regulatory networks, or biomarker discovery which require the interrogation of specific sets of well-characterized genes. In these cases, sequencing the entire transcriptome may be adding unnecessary project costs. To increase throughput and minimize sequencing costs, the development of a targeted gene enrichment method is required.
Here, we extend our whole transcriptome (WT) split-pool combinatorial barcoding technology to enrich a subset of genes in 16 libraries representing hundreds of thousands of human bone marrow mononuclear cells (BMMCs) from three acute myeloid leukemia (AML) and one acute lymphocytic leukemia (ALL) donors. We used our 1,000 immune gene panel to enrich genes representing canonical immune cell markers and pathways. Our method increased the percent of reads on target from as low as 7% in the whole transcriptome libraries to 75% in the targeted libraries. Furthermore, despite a nearly ten-fold reduction in sequencing reads between unenriched and enriched libraries, the resulting clustering yielded high concordance of cell type identities and preserved leukemia-specific signatures such as FLT3, MKI67, and CD19.
Overall, we demonstrate our modular enrichment strategy preserves biological structure and allows for deep characterization of gene signatures in health and disease. We envision our approach will enable researchers to simultaneously reduce sequencing costs while drastically scaling up the number of cells and samples across experiments.
Novel Workflow for Activation and Expansion of T Cells Using Microbubble Technology
Authors: Ellis Jason S , Barrett Crisha B , Snow Tiffany , Roussey Jon , Carey Maureen , Ku Chia , Grimley Edward , Wegner Casey J , McNaughton Brandon H 
Published: August 2023

Abstract
Chimeric antigen receptor (CAR) T cell therapy was developed to address the significant hurdles to cancer treatment. In the US, one in four deaths is due to cancer, with treatment costs totaling over $200 billion a year. Traditional treatments, such as chemotherapy and radiation therapy have serious harmful side effects, often with limited success. Despite the immense hurdles to producing a cost effective, efficient, and scalable workflow for manufacturing CAR T cells, thousands s of CAR T cell therapy clinical trials have been initiated around the world. Traditional CAR T cell workflows often result in T cell exhaustion, limiting the effectiveness of the cells produced. At Akadeum, we set out to create a simple workflow to improve researchers’ ability to develop custom cell therapies. Using Akadeum’s microbubbles, we developed a process that combines selection, activation, and expansion into a single workflow. Because of the natural buoyancy of the microbubbles, activation and expansion take place at the top of the culture. As the T cells divide, daughter cells fall to the bottom of the well away from the activation signals. This prevents overstimulation and the resulting exhaustion phenotype that occurs with the industry-standard activation methods (25–30% PD-1+ with Akadeum method compared to &gt;90% with traditional workflow). This method effectively activates T cells, with significant upregulation of standard activation markers, greater than 20-fold expansion, and desirable effector cell ratios. These cells are also highly transducible. Taken together, these data show that Akadeum’s microbubble-based workflow provides all of the critical aspects of CAR T cell generation while eliminating many of the most significant hurdles.
A new automatic gating algorithm replicates expert manual gating of immune cell subsets
Authors: Weit Nicole Yvonne , Bowers Rita , Grazia Giulia 
Published: August 2023

Abstract
Machine learning is increasingly used for the analysis of flow cytometric immunophenotyping data. Many analysis pipelines leverage unsupervised approaches such as dimensionality reduction or clustering, which may lead to populations characterized differently from expert definition.
Here we show a new, supervised algorithm for population identification that faithfully replicates the expert manual gating strategy.
Peripheral blood samples were drawn from 41 different donors, stained with a dry, unitized B cell panel and acquired on two cytometer units. A manual gating strategy was established, and an automatic model trained on a subset of samples (n=31 donors, split into training, validation and test set) from this study. The performance of the automatic model was evaluated on the 10 unused samples.
The automatic identification of populations with differences in median abundance (lymphocytes, CD19+ B-cells and CD19+ CD10+ cells) showed a high level of correlation and lower variability compared to manual gating. The supervised approach presented here retains the gating hierarchy, in contrast to the unsupervised clustering performed by FlowSOM, which splits cells into exclusive metaclusters, leading to metacluster assignments significantly different from expert gating.
By automating the identification of populations, assay reproducibility can be increased by removing inter-operator variability. We show that a supervised machine learning tool that supports user-defined panels and gating strategies can reproduce expert analysis, reduce variability of flow cytometry data analysis, and can provide better fit-for-purpose compared to an unsupervised clustering approach.
Leveraging assay standardization with DURAClone dry antibody panels on six different flow cytometry platforms
Authors: Bowers Rita , Grazia Giulia , Kapinsky Michael , Lacaille Vashti , Schneider Laura 
Published: August 2023

Abstract
The blessing of Flow Cytometry’s versatility and flexibility comes with the curse of human operator dependence, variable or unstable reagent performances and an infinite number of possible assay designs. Aiming at mitigation of the risk of poor data comparability, DURAClone pre-formulated dry antibody panels provide standardized, technically validated assays in single test-unitized format, devoid of antibody pipetting. To demonstrate that DURAClone standardization can be leveraged on different hardware architectures, including different principles for signal detection and processing, we used six different flow cytometry platforms to acquire DURAClone-stained samples. Due to logistic limitations excluding the use of identical samples in all three different participating institutions, only a qualitative comparison was feasible. Here, we show example data from DURAClone-stained samples acquired on a BD Biosciences FACSCanto II, FACSLyric or FACSymphony flow cytometer, a NovoCyte Quanteon flow cytometer, a BioRad ZE5 flow cytometer or a Cytek Aurora flow cytometer. The presented data confirms expected staining patterns obtained with selected DURAClone antibody panels, irrespective of the flow cytometer used to acquire the data.
Insufficient epitope specific T cell clones is responsible for impaired cellular immunity to inactivated SARS-CoV-2 vaccine in the elderly
Authors: Xiao Chanchan , Chen Guobing 
Published: August 2023

Abstract
Aging is a critical risk factor for SARS-CoV-2 vaccine efficacy. Although low humoral immunity in the elderly was observed in mRNA and recombinant protein vaccination, the immune responses to inactivated vaccine in the elderly, and the underlying mechanisms of differences to young, if any, are still unclear. Here, we studied a cohort of 121 young (18–30 years old) and 48 old (60–85 years old) donors vaccinated with inactivated SARS-CoV-2 and demonstrate that the neutralizing antibody response is slower in the elderly, but eventually reached the similar level to day 7 post-vaccination in young. We identified a range of epitopes targeted by CD8 T cells that were underrepresented in immune responses in the old group, especially when measured with tetramers derived from the 13 major SARS-CoV-2 variants. Comparison of the transcriptomes of B, CD4 and CD8 T cells from pre- and post-vaccinated young and elderly revealed genes potentially responsible for low immune responses in elderly, functionally related to antigen processing and presentation. We further built an epitope specific TCR repertoire database using single-cell RNA and TCR sequencing. Statistical and machine-learning based analyses demonstrated that SARS-CoV-2 epitope specific CD8 T cell clones were significantly lower in the old than young, and failed to be boosted after vaccination in elderly. Comparison of BCRs and TCRs from pre- and post-vaccinated young and elderly individuals revealed inadequate receptor repertoire size and diversity might be responsible for low immune response in the elderly. Together, the altered immune cell function and the attenuated antigen specific TCR repertoire were responsible for the impaired CD8 T cell immune response in the elderly.
This work was supported by grants from the National Key Research and Development Program of China (2018YFC2002003 to G.C., 2021YFC2009400 to G.Z.), the Natural Science Foundation of China (U1801285 and 92169102 to G.C., 81971301 and 32050410285 to O.J.L., 82201954 to Z.R.)
Antibody response of Comirnaty vaccine in different immunocompromised groups during the COVID-19 pandemic
Authors: Togoo Khongorzul , Batmunkh Burenjargal , Tsogtbayar Nomin-Erdene , Batmunkh Ulziisaikhan , Selenge Enkhtuya , Togtokhbaatar Khuselt-Od , Boldbaatar Gantuya , Jagdagsuren Davaalkham , Gunchin Batbaatar , Sandag Tsogtsaikhan 
Published: August 2023

Abstract
A nationwide vaccination program was initiated on February 23, 2021, in Mongolia by 4 types of vaccines for COVID-19 were offered voluntarily. People at increased risk of severe disease were vaccinated with Comirnaty. In this study, we aimed to compare specific antibody responses after the second dose of the Comirnaty vaccine in different immunocompromised groups. This prospective cohort study was conducted from March–April to July–August of 2021. 225 vaccinees presented an immunocompromised population examined for anti-SARS-CoV-2 RBD-IgG (ELISA, Proteintech, USA) before the first dose of vaccine and RBD-IgG antibodies were measured 14 – 21 days after the second dose of vaccine. An increase of antibody titer more than 3-fold was considered a positive antibody response after vaccination. We could attend to the after the second dose of examination 172 (76.4%) of 225 vaccinees. Antibody response to COVID-19 vaccines was detected in 164 (95.1%) vaccinees that demonstrated positive antibody response. A positive response was found in all (100.0%) immunocompromised (19 patients received anti-cancer chemotherapy, 90 patients received corticosteroids, 63 HIV-infected patients) groups. The mean titer of RBD-IgG antibodies in anti-cancer chemotherapy (185.1±49.1ng/mL, CI95: 161.4–208.8) and corticosteroids group (161.6±70.3 ng/mL, CI95: 147.1–176.2) was significantly higher compared to HIV-infected group (98.8±32.6 ng/mL, CI95: 90.6–107.0). A significant impact on antibody response after the second dose of Comirnaty COVID-19 vaccination was associated with the immunocompromised condition. The study was funded by the Ministry of Health of Mongolia.
None
Understanding Mechanisms of Adjuvancy in Muscle by mRNA/Lipid Nanoparticles
Authors: Majumdar Devdoot , Dowell William , Languon Sylvester , Miller Zachary , Dearborn Jake 
Published: August 2023

Abstract
Vaccines utilizing encapsulated mRNA within lipid nanoparticles (LNPs) have demonstrated unprecedented efficacy in clinical studies of mRNA-1273 and BNT162b2, two prominent vaccines developed against the S protein of SARS-CoV-2. Despite several Phase III trials offering deep clinical intuition about vaccine efficacy, the musculo-immune basis for elicitation of a strong germinal center response from mRNA/LNP vaccine remains unclear. Here, we characterize the trajectory of the immune response to an mRNA lipid nanoparticle vaccine, encapsulated in sm-102 (Moderna). Furthermore, we find that intramuscular administration of empty LNPs elicits a strong neutrophil and dendritic response within 24h. Additionally, we show that skeletal muscle may play a role in the early immune response to mRNA LNPs. Inflammatory LNPs thus contribute to the well-established role of RNA immunogenicity. Using a reporter of transduction, we show that both &gt;10% of muscle-resident myeloid and lymphoid cells are directly transduced with mRNA vaccines, and that these cells play a crucial role in the downstream germinal center response. Transcriptomic measurements of muscle tissue reveal a direct role role of ionizable lipid operating as an adjuvant to induce this response. Using in vitro and molecular tools, these studies advance our understanding of the cellular and molecular basis for LNP-mediated inflammation and adjuvancy underpinning the exceptional clinical efficacy of mRNA/LNP vaccination.
Detection of envelope-dimer epitope-like broadly protective antibodies in dengue-immune children in the Philippines following vaccination and natural infection
Authors: Mpingabo Patrick Ilunga , Ylade Michelle , Crisostomo Ma Vinna , Thiono Devina , Daag Jedas Veronica , Coello Ana E , Rodriguez Guillermo R , Lowman Kelsey , Firdous Saba , Aogo Rosie , Odio Camila , White Laura , de Silva Aravinda , Deen Jacqueline , Katzelnick Leah C 
Published: August 2023

Abstract
The dengue vaccine, Dengvaxia, has been described as eliciting antibody-dependent enhancement (ADE) in dengue-naive individuals. There is an urgent need to develop an effective dengue virus (DENV) vaccine that elicits protective, cross-reactive (CR) neutralizing antibodies (NAbs), which are capable of neutralizing DENV1–4 without triggering ADE such as EDE Abs.
Here, we investigated the CRNAbs profile following DENV natural infection versus vaccination in children with prior DENV infection (n=2,996) from a longitudinal vaccine (study) cohort in Cebu, Philippines. In total, 1,214 of these children remained unvaccinated while 1,782 children received a single dose of Dengvaxia in June of 2017 during a mass vaccination campaign. Serum samples were collected one month before and one year after the vaccine campaign.
To measure baseline status and change in CRNAbs level following vaccination versus natural infection, we ran Focus Reduction Neutralizing Test (FRNT) using mature DENV1–4 virions and Blockade of binding (B0B) assay against EDE Abs on a random subset of these DENV-immune children’s samples (n=231). Furthermore, we also measured IgG binding against the DENV1–4 E protein monomer and DENV2 E protein dimer.
Both vaccinated and naturally infected groups elicit CRNAbs that recognize the quaternary epitopes of EDE antibodies. We also see a significant increase in antibody response in both groups, but CRNAbs had a stronger correlation to mature FRNT in naturally infected compared to vaccinated populations, indicating differences in antibody quality. These results highlight the presence of EDE-like broadly NAbs that could possibly protect against the four DENV serotypes and provide an insight for future vaccine candidates.
This work was supported by the Intramural Research Program (DIR) of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, the Philippine Department of Health and the University of North Carolina (PO1: P01AI106695).
PD-1 immune checkpoint blockade can synergize with PSGL-1 inhibition to reinvigorate exhausted T cells
Authors: Viramontes Karla M , Neubert Emily N , DeRogatis Julia M , Henriquez Monique , Tinoco Roberto 
Published: August 2023

Abstract
P-selectin glycoprotein ligand-1 (PSGL-1) is an immune checkpoint regulator that is highly expressed on T cells. PSGL-1- deficient mice infected with chronic lymphocytic choriomeningitis virus (LCMV) were shown to control chronic viral infection due to increased effector function by anti-viral T cells. However, the cell-intrinsic role of PSGL-1 expression on CD4+ and CD8+ T cell exhaustion is not fully known. Using adoptive transfers of WT or PSGL-1-deficient TCR transgenic CD4+ and CD8+ T cells in mice infected with chronic LCMV, we characterized the differentiation and effector response. We found increased expansion and effector function by PSGL-1-deficient T cells early during infection, however, at later stages of infection, PSGL-1-deficient T cells were functionally exhausted. We found that exhausted CD4+ and CD8+ PSGL-1-deficient T cells were reinvigorated more effectively than WT cells after PD-L1 blockade due to their increased proliferation, cytokine production, and accumulation. We also observed increased responses of CD8+PSGL-1-deficient T cells in melanoma tumors. Our findings showed that PSGL-1 expression was required for the maintenance of both CD8+ and CD4+ exhausted T cells and that PSGL-1 deletion synergized with PD-1 blockade to reinvigorate these cells. These findings highlight an important cell-intrinsic role for PSGL-1 expression in the maintenance and reinvigoration of exhausted T cells.
T32 AI 141346 R01 AI13723
B cell dysfunction during aging is not dependent on stem cell aging
Authors: Greer Braxton D , Maul Robert W , Gearhart Patricia J 
Published: August 2023

Abstract
Humoral immune responses to vaccination are a critical component of protection from infectious diseases. Older humans simultaneously have reduced antibody production in response to vaccination and an increased burden of mortality when infected with viral respiratory infections such as influenza or SARS-CoV-2. Therefore, it is critical to understand molecular drivers of immunosenescence in order to develop vaccination strategies that better protect the elderly. By adoptively transferring naïve B cells from old or young mice into young B cell-depleted recipients, our lab has characterized the germinal center dynamics and antibody production in response to a model antigen, nitrophenyl-chicken gamma globulin (NP-CGG). B cells from young mice were found to be antigen specific in the germinal center and produced specific anti-NP antibodies. B cells from old mice failed to produce anti-NP antibodies and had a large proportion of non-specific germinal center B cells. We sought to uncover the origin of this age-related deficiency. We hypothesized that age-induced changes in hematopoietic stem cells (HSCs) might be passed on to mature B cells and disrupt their function. We transplanted HSCs from young or old donors into young, irradiated recipients and analyzed the ability of the progeny B cells to mount a specific antibody response. To our surprise, HSC age did not encode B cell function, and B cells from both HSC donor ages could mount an NP-specific response, implicating an alternative source of aging dysfunction. Further study into the cause of age-related B cell dysfunction will guide the development of vaccine additives that improve protective immunity in the elderly.
Supported by NIA intramural funding.
Heme augments proliferation and epigenetic programming during B cell differentiation
Authors: Padilla-Quirarte Herbey O , Kania Anna , Hicks Sakeenah , Maurer Jeffrey , Boss Jeremy M , Scharer Christopher D 
Published: August 2023

Abstract
Heme is an iron-containing porphyrin ring that is a key part of the cytochrome proteins that function in the electron transport chain and facilitate OXPHOS metabolism. Recently, our group found that many iron-metabolism related genes are highly expressed in B cells, particularly in memory B cells (MBC) in both mice and humans. In ex vivo cultures, naïve (nB) and MBC respond to heme addition by augmenting the frequency of plasmablast formation. In this work, we further characterized the role of heme in augmenting B cell differentiation and function. To begin addressing the question of how heme modulates B cell differentiation, proliferation, or both, we first conducted ex vivo cultures of CellTraceYellow (CTY)-labeled B cells stimulated with CD40, IL-4 and IL-5, treated with and without heme. We found that the first division occurs within 28 hours post-stimulation for nB cells and nB and MBC that were treated with heme divide earlier than those that were treated with vehicle.. When the number of cells in each division was analyzed, we found more cells in later divisions in heme-treated than in vehicle-treated ones, suggesting that heme is important for B cell proliferation. To further characterize the molecular effects of heme on B cell differentiation we preformed RNA-seq and ATAC-seq on activated B cells and plasmablasts. The addition of heme augmented both the accessible chromatin and gene expression of hundreds of genes leading to differences surrounding key metabolic and differentiation regulators. These data further our understanding for how heme modulates B cell differentiation and promotes the formation of antibody secreting plasmablasts.
PAD4-dependent neutrophil extracellular traps dagame intestinal barrier integrity through histone protein components
Authors: Chiang Hao-Sen 
Published: August 2023

Abstract
Infiltrating neutrophils and neutrophil extracellular traps (NETs) accumulate in the inflamed epithelium to induce mucosal inflammation in patients with inflammatory bowel diseases. The formation of NETs is initiated by peptidyl arginine deiminase 4 (PAD4), which facilitates NET generation through chromatin decondensation. We recently demonstrated that NETs impair intestinal epithelium integrity, permeability, and viability in mice with colitis. In addition, disruption of NET formation by administering DNase I or PAD4 inhibitors attenuates intestinal inflammation in colitic mice. However, the effect of genetic loss on PAD4-mediated NET formation and intestinal inflammation has not been determined in chemical-induced colitis models. Moreover, the specific NET components responsible for the damage and impaired barrier integrity in the intestinal epithelium are also unknown.
Here we demonstrated that mice lacking PAD4 failed to produce NETs in the intestine in response to dextran sulfate sodium (DSS)-induced colitis. This impairment in PAD4-deficient mice ameliorated intestinal inflammation, barrier function, and integrity. Mechanistically, NETs, particularly the histone components, altered the integrity of junctional proteins and induced cytotoxicity, resulting in increased gut epithelium permeability. In contrast, inhibition of histone activity in NETs restored the expression and localization of junctional proteins and cell viability, thus enhancing intestinal barrier functions. Collectively, our findings indicate that extracellular histones of NETs have a detrimental role in regulating intestinal barrier function in Caco-2 monolayers and in vivo.