bluuebunny/crossref_metadata_2025_split huggingface dataset:
SIMON, a powerful AI software accelerating discovery of human immune memory responses to viruses
Authors: Tomic Adriana , Tomic Ivan , Skelly Donal T , Ogbe Ane , O’Connor Daniel , Pollard Andrew J , Dold Christina , Barnes Eleanor , Klenerman Paul , Dunachie Susanna , Davis Mark M , Consortium Pitch 
Published: August 2023

Abstract
Influenza and SARS-CoV-2 are among the greatest viral threats to human health causing the 20th and 21st centuries’ most significant pandemics. These viruses have caused millions of deaths worldwide and continue to pose a significant risk to global health. To effectively fight against these viruses, it is essential to understand the complexity behind the generation of immune memory responses.
The traditional analytical methods are often inadequate for capturing the complexity of the immune responses. To address this challenge, we have developed SIMON, a new AI software that can perform integrative multi-omics analysis to study human immune memory responses. With an easy-to-use graphical user interface, standardized pipelines, automated approach for machine learning testing over 180 algorithms, and other statistical analysis methods, SIMON helps to identify crucial patterns in biomedical data. We have demonstrated the accuracy, ease of use, and power of our software on various biomedical datasets, gaining valuable insights into the mechanisms underlying immunity to SARS-CoV-2 and influenza. This knowledge can be used to develop more effective vaccines and help researchers better understand the long-term impact of SARS-CoV-2 or influenza viruses on the immune system. By predicting the immune memory responses, SIMON has the potential to accelerate the development of vaccines that provide long-term protection against these pandemic viruses. Overall, SIMON is a powerful software platform for data mining that facilitates pattern recognition and knowledge extraction from high-dimensional biomedical data. It has the potential to greatly accelerate discovery in human immunology and biomedicine.
Phenotyping complex human B cell compartments using high parameter spectral flow cytometry and scRNA-seq with parallel CITE-seq analyses
Authors: Meinhardt Nathan , Dittel Bonnie 
Published: August 2023

Abstract
The human B cell compartment is very complex, consisting of numerous B cell subsets. Classifying this heterogeneous population into discrete subsets based on surface phenotype alone remains a major challenge in the field of B cell biology. Our lab has discovered a novel mouse IgM+ IgDlow/− B cell population that induces Treg proliferation in a GITRL-dependent manner, making it a potentially significant therapeutic target for autoimmune diseases. But due to their rarity and lack of specific cell surface identifiers, a more definitive phenotype is required to track this B cell subset in humans. The objective of these studies is to characterize human IgDlow/− B cells from peripheral blood using both high parameter spectral flow cytometry and scRNA-seq with parallel CITE-seq analyses. Spectral flow cytometry has the major advantage over its conventional predecessor by allowing for the construction of large parameter panels that are not limited by standard compensation. Here, we have adapted a standardized gating scheme (CD27 vs. IgD and CD24 vs. CD38) that captures all the major canonical human B cell subsets. Furthermore, we have used analysis plugins in FlowJo (Phenograph and FlowSOM) that predict multiple discrete B cell subpopulations within the IgDlow/− phenotype. Additionally, we have analyzed human B cells (CD19+ IgM+ IgDlow/−) using scRNA-seq combined with CITE-seq. This approach allowed us to analyze both the cell surface protein expression of CD27, CD21, CD24, and CD38 combined with the transcriptome at the single-cell level. Our analysis has revealed four individual subclusters within this populationthat can be validated by flow cytometry, showcasing the power of combining these two cutting-edge analytical tools.
HLA-PopSeq: High throughput, multiplexed six-locus Human Leukocyte Antigen typing for population-scale T cell immune profiling using rapid long-read nanopore sequencing
Authors: Kask Oliver , Knappenberger Mark , Lee Heewook , Van Stone Alicia , Anderson Karen 
Published: August 2023

Abstract
A major technical hurdle for T cell immune profiling is the time and cost to accurately genotype the Human Leukocyte Antigen (HLA) loci from peripheral blood. Here, we developed a rapid, highly multiplexed approach for HLA typing using RNA from &lt;100,000 peripheral blood mononuclear cells with the Oxford Nanopore Technology (ONT) Minion sequencer. This method uses selective reverse transcription of mRNA of six HLA loci (A,B,C, DRB1, DQB1, DPB1), followed by PCR amplification. The individual amplified HLA cDNA was multiplexed in a single sequencing pool using primers with unique molecular identifiers, designed to permit sequencing errors for enhanced data capture. Pooled HLA amplicons were sequenced using the ONT Minion MK1B and R10.4 flowcells, with sequence Q scores&gt; 20. Total RNA was extracted from PBMC samples from 12 individuals, reverse transcribed and amplified using the designed HLA loci specific primers. The pooled, amplified cDNA was then sequenced for 16 hours on the ONT Minion sequencer. The resulting sequencing data was analyzed and an average depth of coverage of 6000x was observed per sample. An average per loci depth of coverage of 1000x was observed. This method is designed to permit rapid (&lt;24h), low-cost, portable HLA sequencing for T cell immune monitoring and epitope identification for immunologic studies.
Arizona Piper Foundation
Using nanoparticles as artificial antigen presenting cells to mobilize CD4 T cells for immunotherapy
Authors: Kang Si-Sim , Isser Ariel , Schneck Jonathan P 
Published: August 2023

Abstract
CD4 T cell-based adoptive cell transfer (ACT) therapies have clinical success across multiple cancer types, including metastatic melanoma and epithelial cancer. One of the challenges of ACT therapy is finding methods to expand tumor antigen-specific T cells ex vivo. Therefore, engineering platforms for antigen-specific T cell expansion are critical since they provide greater control over studying T cell biology and have significant translational relevance.
We developed a nanoscale platform that expands murine and human antigen-specific T cells in vitro. This artificial antigen-presenting cells (aAPCs) platform provides T cells with two critical signals, MHC class II and anti-CD28, for T cell activation, which permits the activation of cognate antigen-specific CD4 T cells ex vivo. We isolate human CD4 T cells from PBMCs of healthy donors who are HLA DP4+. We then stimulate CD4 T cells with aAPCs in media with inflammatory cytokine and access the specificity and effector function through tetramer staining, cytokine release assays, and killing assays.
Co-culturing aAPCs and human CD4 T cells significantly increase the frequency of cognate antigen-specific CD4 T cells. Specifically, we expanded tetanus toxin-specific HLA-DP4+ CD4 T cells from healthy human PBMCs with aAPC. By the end of the expansion, the antigen-specific expanded from less than 1% to over 40% by day 21. In addition, the resulting antigen-specific cells display high levels of effector cytokine production, including TNF-a, IFN-g, IL-2, and granzyme B. Consistently, aAPC-activated CD4 T cells demonstrate robust lytic capacity in vitro and in vivo. These data show that aAPC can expand antigen-specific human cytotoxic CD4 T cells from a rare precursor population.
High-throughput workflow leveraging spectral flow cytometry and CITE-seq to characterize human bone marrow across race and gender
Authors: Song Baobao , Zhang Xuan , Bennett Sierra , Ma Siyuan , Ferchen Kyle , Salomonis Nathan , Grimes H Leighton 
Published: August 2023

Abstract
Characterizing human bone marrow is challenging due to heterogeneity of cell types, and the rarity of some populations. To address this, we developed a workflow that bridges CITE seq and flow cytometry. First, we rigorously titrated 277 TotalSeq-A antibodies (BioLegend) using Laminar Wash technology (Curiox Biosystems) to efficiently wash unbound antibodies in a hands-free manner and improve reproducibility. We resolved the utility of 133 TotalSeq-A antibodies (and 5 isotype controls) for CITE-seq of primary human bone marrow samples. We describe the application of these 133 markers in CITE seq of primary marrow samples (varying sex and race) to create a human bone marrow atlas with both transcriptional and antibody-based markers for over 70 cell states. Notably, we also discovered race-based differences in molecular markers and population frequencies. Next, we exploited a machine learning tool (pyInfinityFlow) to validate these molecular/informatics findings. We used a 22-color spectral flow panel tailored to human bone marrow (developed by Cytek Biosciences) as the backbone, then incorporated 111 PE-conjugated “Infinity markers” guided by the CITE-seq results. The analysis resolved a single final FCS file to co-visualize 133 markers, and provided concordance between CITE-seq values measured by sequencing and flow values measured by fluorescence intensity for marrow populations. In summary, we present a streamlined workflow widely adaptable to bone marrow disease studies and transferrable to other tissues. We deliver an atlas-level high-resolution reference with transcriptome-defined populations and immunophenotyping, enabled by both sequencing and spectral flow cytometry.
NIH RC2DK122376-03
Accurate enumeration of leukocyte subsets in peripheral blood with a 15-color immunoprofiling panel on a Cytek® spectral flow cytometer
Authors: Fukazawa Yoshinori , Edinger Mark , Fleming Chris , Amador Ivette , Chen Jingyi , Deng Jun , Wang Alice 
Published: August 2023

Abstract
A white blood cell (WBC) differential is routinely performed on automated hematology analyzers as a general health check to evaluate immune status of patients. The recent advances in high parameter flow cytometry, especially full spectral flow cytometry, enable the analysis of more comprehensive leukocyte subsets to meet the increased demands in clinical research. We designed a 15-color, single-tube panel to identify and enumerate all major subsets of leukocytes on a Cytek full spectral flow cytometer.
Human peripheral blood collected in K2EDTA, Heparin, ACD and Cyto-chex blood tubes were stained with 15 cFluor® conjugated mAbs with a Lyse No Wash method and analyzed on a Cytek spectral flow cytometer. The performance of this assay was evaluated side by side with BD Multitest™ 6-color TBNK assay on BD FACSCanto II system on 13 healthy donor samples. Assay precision was examined on 12 donor samples in triplicates on three consecutive days.
This 15-color panel identifies more than 20 leukocyte subpopulations: including neutrophils, eosinophils, basophils, lymphocytes, hematopoietic stem cells, monocyte subsets, T cell subsets, B cells, NK cell subsets. The results are comparable to that from BD 6-Color TBNK assay with R2=0.961, 0.986, 0.940, 0.954, and 0.930 for CD3+, CD4+, CD8+ T, B and NK cells respectively. The correlations for all subsets are statistically significant with P &lt; 0.001. The coefficient of variations (CV) of intra assay precision for all cell populations of interest are less than 25%.
Cytek’s single-tube 15-color pan leukocyte panel demonstrated an effective, high sensitivity flow cytometry approach that can be used for monitoring immune cell subsets in peripheral blood in discovery and translational research.
In vivo imaging system for cellular level visualization of immune responses in various animal models
Authors: Kim Alex Jin Woong , Kim HyunSeok , Akyildiz Kubra 
Published: August 2023

Abstract
Immune cells have a variety of functions to defend against pathogens under certain circumstances. It is not until recently, However, that these miscellaneous responses can be observed in vivo. Herein, we optimized some solutions of researchers keeping track of the changes of the immune response by utilizing the IVM-C intravital imaging system and in vivo fluorescence labeling for target cells. It is possible to successfully acquire a crystal-clear cellular level images of immune cells in the different animal model including RNA adjuvant-injected mouse and sepsis-induced one by using intravital microscope, respectively. In case of the RNA adjuvant injection, it is obvious that retention of the antigen presenting cells in draining lymph node is on the rise to promote T/B cell activation. A dramatic increase in the number of the infiltrated dendritic cells into the organ was observed at the same time. Moreover, Pathogenesis of sepsis by monitoring of pulmonary capillary circulation was investigated. In order to visualize pulmonary microcirculation, we’ve established a pulmonary imaging window which makes it feasible to image the real-time dynamics of rapidly circulating neutrophils in live mouse. We found out that many clusters of neutrophils aggregates in the pulmonary vasculature or, even in the arteriole of the sepsis-induced mouse, thereby disturbing the microcirculation of that region and generating dead space in the pulmonary structure. Taking everything into account, we make certain it is appropriate to utilize the intravital imaging method for cellular level visualization of the immune system under various pathological conditions. we believe that it could be an invaluable tool for a comprehensive understanding of immunology.
A single-tube, 20-color panel effectively detects aberrant cells in acute myeloid leukemia
Authors: Chang Qing , Edinger Mark , Chen Jingyi , Deng Jun 
Published: August 2023

Abstract

Objective
Multiparameter flow cytometry is widely and routinely used in acute myeloid leukemia (AML) diagnosis and the residual disease detection and monitoring. We have developed a single-tube, 20-color panel for AML analysis that uses the Cytek® spectral flow cytometer with increased reagent and sample efficiency.

Methods
Blood and bone marrow samples were stained with the 20-color panel, acquired, and analyzed using SpectroFlo® software. The resolution of each marker was compared between single stain versus the 20-color full stain on the same samples. The analytical precision for cell populations of interest and antibody cocktail stability were assessed. The limit of detection (LOD) and lower limit of quantification (LLOQ) of the 20-color assay were determined using limiting dilution method.

Results
The resolution of each marker in the fully stained panel was comparable to that in the single-color stained samples. Normal and aberrant myeloid cell populations were clearly identified and the coefficient of variation (CV) of cell percentage for defined populations in replicate runs were all less than 25%. The antibody cocktail was stable in the fridge for at least 14 days. Our data showed that the single-tube, 20-color assay easily achieved 0.01% on residual aberrant cells in AML, higher than the minimum required detection sensitivity of 0.1%.

Conclusions
Cytek’s single-tube, 20-color panel demonstrated an effective, high sensitivity flow cytometry approach that can be used for AML testing, including identifying and characterizing normal and aberrant cells, immunophenotypic classification and minimal residual disease evaluation in translation research.
Rational antigen design for cell based immunological assay systems in different immunological subjects
Authors: Strecker Kevin , Ettischer-Schmid Nicole , Preyer Rosemarie 
Published: August 2023

Abstract
Cell based immunological assay systems are widely used in research as well as routine diagnostics in these days to gain better and more detailed information compared to classical assay formats. Despite the less varying framework of well-established standard assay formats like ELISA, Flow Cytometry or EliSpot Assays, the antigens used for stimulation are undergoing dynamic changes as new applications are needed to manage complex emerging immunological challenges.
Consequently, the most important component in such assay systems are the antigens used for stimulation. There are several methods to generate different types of stimulants which all have their own inherent advantages and disadvantages depending on their intended use and immunological problem being addressed. It is mandatory to ensure that the strategy used for the choice of a suitable antigen type is compatible with the used assay format and cell type analysed. Furthermore, the quality and design of those antigens are main factors influencing a clinically sufficient sensitivity and specificity of the final assay system. Depending on the desired clinical outcome of a given assay system, not all types of antigens fulfil the necessary requirements to gain sufficient results. Here we want to give a comprehensive overview of the most common available methods in antigen design and give advice when to use which kind of antigen type depending on the specific application. We will show the general tasks to accomplish during the process of designing new antigens used as stimuli in cell based immunological assay systems.
Spatially resolved whole-transcriptome analysis with simultaneous highly multiplexed immune cell epitope detection in multiple cancer tissues
Authors: Gupta Anushka , Williams Stephen , Gutgasell Lauren , Veire Benton , Santiago Ace , Singh Hardeep , Chan Rena , Hermes Alex , Kamath Govinda , Patel Anuj , Russel Spontaneous , Sukovich David 
Published: August 2023

Abstract
The tumor microenvironment is composed of highly heterogeneous niches, often with varying degrees of immune infiltration. The spatial distribution of immune cells with respect to malignant cells can directly impact patient prognosis and overall survival outcomes. The Visium CytAssist Spatial Gene Expression assay uses a whole transcriptome probe-based approach, termed RTL, to detect and quantify mRNA expression with spatial context. Although examination of the tumor microenvironment with an RTL-based spatial assay can provide significant transcriptomic information concerning regions of interest, immune cells frequently have extremely low mRNA expression levels and can be difficult to detect. The use of antibody-conjugated probes specific to immune cell epitopes, which are highly expressed, can enhance data recovered from these tumor samples, enabling spatially accurate detection of immune populations. The Visium CytAssist Spatial Proteogenomic Solution enables identification of immune-specific epitopes via antibody-conjugated probes from the same tissue slide used for transcriptomic analysis. Using the CytAssist workflow, we showcase the ability to comprehensively resolve immune cells associated with multiple immune and tumor tissues, including an array of human breast cancer punches. Spatial expression patterns of immune markers map back to distinct morphological features within the samples, allowing identification of differentially-expressed genes associated with those areas. Overall, these data highlight the value of Visium CytAssist Spatial Proteogenomic Solution in immuno-oncology studies, through the integration of spatially resolved transcriptomic and immune cell marker data.
The BD Rhapsody&[trade] HT Xpress System supports dynamic and high-throughput single-cell capture enabling a seamless workflow for analysis of cellular immune responses
Authors: Shi Samuel , Baracho Gisele V , Song Hye-Won , Acob Ricelle A , Ayer Aruna , Widmann Stephanie J , Tyznik Aaron J 
Published: August 2023

Abstract
The BD Rhapsody™ Single-Cell Analysis System is an innovative platform that aids high-throughput sequencing of mRNA and proteins in thousands of single cells. Here we present a new eight-lane cartridge design concept, the BD Rhapsody™ HT Xpress System. This novel device architecture can reduce both hands-on and assay time by enabling the users to simultaneously load multiple cell samples while preserving the flexibility to use one lane at a time. To demonstrate the increased throughput capabilities and workflow flexibility of the system, we measured in vitro T cell activation over time. To do this, peripheral blood mononuclear cells from four different subjects were prepared for cell culture in IL-2 or IL-2/anti-CD3/anti-CD28. After one day in culture, cell samples from each culture condition were barcoded using the BD® Single-Cell Multiplexing Kit. Cells from the same subject were pooled and stained with the BD® AbSeq Immune Discovery Panel. Pooled cells from each subject were loaded into the cartridge (one subject per lane, four lanes total). Remaining cells were cultured for another day and similarly barcoded, stained and loaded into the other four lanes of the cartridge. Eight different next generation sequencing libraries were generated and analyzed with the BD Rhapsody™ Analysis Pipeline and SeqGeq™ Software. This strategy enabled us to assess dynamic changes in both T cell surface protein profiles and transcriptome during cell activation while using a streamlined workflow for concomitant analysis of multiple samples.
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Simultaneous detection and characterization of antigen-specific B cells and CD4+ and CD8+ T cell responses upon natural infection and vaccination
Authors: Inekci Dilek , Hansen Bjarke Endel , Brix Liselotte 
Published: August 2023

Abstract
An important lesson learned from the COVID-19 pandemic is the need for a more comprehensive assessment of the immune status upon a natural infection and vaccination. In addition to measuring antibody titers, monitoring the status of antigen-specific B and T cell must be taken into consideration to gain a more complete understanding of the elicited immune response and its durability.
The objective of this study was to develop an immune monitoring assay based on the Dextramer® technology for simultaneous monitoring of antigen-specific B and T cells (CD4 and CD8). The assay can be applied in combination with flow cytometry and in single cell RNA-seq analysis.
The assay builds on Dextramer® reagents displaying full-length spike protein (specific B cell assessment) or MHC-peptide complexes displaying virus peptides (detecting specific CD8 or CD4 T cells).
The assay was evaluated by longitudinal assessment of immunity in individuals vaccinated against SARS-CoV-2 by combining the assay with flow cytometry (n=5) and the Chromium 10× RNA-seq platform (n=2). The latter allows an in-depth view of the immune status by simultaneous investigation of the transcriptome, surface proteins and receptor sequences of antigen-specific cells.
Our results show that by using the virus-specific Dextramer® assay we could monitor changes in SARS-CoV-2-specific B- and T-cell responses before and after primary vaccination and booster doses using flow cytometry and single cell RNA-seq analysis. These results show that the Dextramer® assay can aid in understanding the elicitation and persistence of antigen-specific responses. Moreover, the assay can easily be tailored to any antigen-specificity by replacing the ligands coupled to the Dextramer® reagents.