bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single cell multi-omics technologies enable human immunogenomics monitoring
Authors: Ji Xuhuai , Miranda Molly , Suresh Sundari , Bader Cameron Scott , Zoubeidi Adam , Meyer Everett H , Maecker Holden Terry 
Published: August 2023

Abstract
Single-cell RNA-seq (scRNAseq) can investigate transcriptomic or targeted gene expression, including V(D)J and surface proteins to provide insights into mechanism discovering and therapeutic monitoring. Multiple scRNAseq technologies have recently been developed and emerged as a major driver of progress in biomedical research. Here we used three scRNAseq platforms, BD Rhapsody, Fluent PIPseq, and Parse Evercode, to monitor immunogenomics variation of certain immune cells from four hematopoietic stem cell transplant recipients. Our results identified major signature genes as potential biomarkers for stem cell transplantation outcomes. We compared an array of metrics from the three platforms in parallel to assess applications, unique advantages, and cost-effectiveness. We conclude that the BD Rhapsody system is the most comprehensive multi-omics platform for immunogenomics analyses of tens of thousands of cells, while Fluent PIPseq & Pasre Evercode have unique features and potential for larger scale single cell multi-omics profiling with Parse enabling up to one million cells per experiment.
supported by 5P01CA049605-32 (EHM) from NIH, K00CA245728 (CSB) from NCI, and OPP1113682 (HTM) from the Bill and Melinda Gates Foundation.
Characterization and comparison of acridine orange/propidium iodide and acridine/DAPI viability detection methods for cell and gene therapy development
Authors: Huang Yongyang , Patel Samir , Pierce Mackenzie , Lin Bo , Chan Leo 
Published: August 2023

Abstract
Cell viability is one of the critical quality attributes (CQAs) for analyzing and characterizing cellular therapeutic products. The characterization of cell viability is essential to ensure quality, safety and efficacy during cell and gene therapy development. In this work, we characterized and compared two commonly used viability dual-staining methods, acridine orange/propidium iodide (AO/PI) and acridine orange/4′,6-diamidino-2-phenylindole (AO/DAPI) using the Cellaca MX high-throughput cell counter. In general, the AO/DAPI method provided higher viability results than AO/PI, with the measured viability difference between these two methods ranging from 0% to 24%. To identify the sources of the measured viability difference, we tested a list of factors including cell dying process, staining time and initial viability levels. First, we observed a larger viability difference from naturally-dying Jurkat cells compared to the result from heat-killed cells, suggesting that the differences may depend on the cell dying process. Second, we found that AO/PI provided more consistent cell count and viability than AO/DAPI during the first 30 min of staining time. Finally, we showed that measured viability difference might be larger for low-viability, naturally-dying Jurkat cells, as compared to healthy samples. The results from our characterization studies may benefit the selection of viability methods that are fit-for-purpose for analyzing cellular therapeutic products.
Tri-modal single cell (TEA-seq) profiling reveals a novel, epigenetically distinct T cell subset in children and global molecular reprogramming across age
Authors: Gustafson Claire E , Thomson Zachary , He Ziyuan , Swanson Elliot , Henderson Katherine , Pebworth Mark-Phillip , Okada Lauren , Heubeck Alexander , Roll Charles , Hernandez Veronica , Weiss Morgan , Genge Palak , Reading Julian , Giles Josephine , Manne Sasikanth , Dougherty Jeanette , Jasen C J , Greenplate Allison , Becker Lynne , Graybuck Lucas , Vasaikar Suhas , Szeto Gregory Lee , Savage Adam , Speake Cate , Buckner Jane H , Li Xiaojun , Bumol Thomas , Wherry E John , Torgerson Troy , Vella Laura , Henrickson Sarah E , Skene Peter 
Published: August 2023

Abstract
Most single cell analyses of human T cell heterogeneity use a single modality (i.e., RNA or ATAC or protein), with limited ability to deconvolute complex cellular and molecular alterations that can occur during human aging and disease. Here, we use a new trimodal single cell technology (TEA-seq) to elucidate the interplay between the surface proteome, transcriptome, and epigenome in more than 300,000 T cells from healthy children (11–13 yrs) and older adults (55–65 yrs). We uncover that age universally drives molecular reprogramming across all T cell subsets towards a more activated basal state. Naive CD4 T cells, considered relatively resistant to aging, exhibited far more pronounced transcriptional reprogramming than naive CD8 T cells, in which alterations are preferentially driven by compositional shifts in naive-like memory subsets. We further discover a novel naïve-like CD8aa T cell subset lost with age that displays an innate-like transcriptome, a KLF/SP-rich epigenome and high IL-21 receptor expression. These data reveal the complex molecular landscape of the human T cell compartment that may contribute to differential immune responses across age.
Supported by grants from NIH (K01 AG068373 to CEG) and the Allen Institute
Lyophilized tolerogenic polymer particles reverse disease in experimental autoimmune encephalomyelitis while retaining drug properties over time
Authors: Martin Corinne A , Kapnick Senta M , Carey Sean T , Froimchuk Eugene , Ackun-Farmmer Marian A , Jewell Christopher M 
Published: August 2023

Abstract
Autoimmune diseases such as Multiple sclerosis (MS) and diabetes affect more than 50 million people worldwide. We have previously demonstrated that intra-lymph node injection of polymer microparticles (MPs) loaded with myelin antigen (MOG) and a tolerogenic immune cue (rapamycin) protect mice from experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. However, an important aspect for safe translation of new therapeutics is ensuring well-defined and stable drug products through encapsulation, storage, and delivery. Here, we demonstrate that lyophilization and storage at 25C on the scale of months does not impact the tolerogenic capabilities of MOG/Rapa MPs in this system. Using nuclear magnetic resonance spectroscopy (H-NMR) and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), we show that biophysical and biochemical properties of cargo remain consistent between lyophilized and non-lyophilized MPs. Similar proton shifts associated with key structures of rapamycin were observed in drug recovered from reconstituted MPs compared with soluble standard. Likewise, mass spectrometry analyses of antigen confirmed that mass indicators were also consistent between standard and lyophilized MOG. Thus, the structural fidelity of cargo was maintained throughout MP lyophilization and reconstitution. Treatment with lyophilized MPs significantly decreased clinical score compared to freshly-prepared treatment, indicating that lyophilization does not impact the ability of particles to protect against EAE. This work demonstrates the capacity for antigen-specific biomaterial immunotherapies to be stably preserved, supporting potential for clinical applications.
Supported by grants from NIH (R01 AI169686, R01 EB026896)
CD4+ T cell epitope identification de novo
Authors: Zdinak Paul , Grebinoski Stephanie J , Torrey Jessica , Zarate-Martinez Eduardo , Hicks Louise , Ranjan Rashi , Trivedi Nishtha , Arshad Sanya , Anderson Mark S , Vignali Dario A A , Joglekar Alok V 
Published: August 2023

Abstract
CD4+ T cells recognize peptide antigens presented on class II Major Histocompatibility Complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor (TCR) sequences and lack of antigen discovery approaches for MHC-II epitopes has made profiling the specificities of CD4+ T cells challenging. To address this, our lab has expanded our platform of Signaling and Antigen-presenting Bifunctional Receptors (SABRs) to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, generating a readable output. We demonstrate that SABR-IIs libraries presenting endogenous and post-translationally modified epitopes can be used for antigen discovery. Using SABR-II libraries in conjunction with single cell RNA sequencing, we have de-convoluted multiple highly expanded TCRs from pancreatic islets of Non-Obese Diabetic (NOD) mice de novo. We have expanded antigen discovery by incorporating computational TCR similarity prediction metrics followed by experimental validation. Finally, we show that SABR-IIs presenting epitopes in class II HLA alleles can be used for antigen discovery for human CD4+ T cells. Taken together, we have developed a rapid, flexible, scalable, and versatile approach for the de novo identification of CD4+ T cell ligands from single cell RNA sequencing data using experimental and computational approaches. We are continuing to de-convolute the specificity of islet infiltrating CD4+ T cells showing high levels of clonal expansion from both mice and humans with an emphasis on screening theoretical libraries containing hybrid insulin peptides.
Supported by grants from NIDDK (1R03DK127447), dkNET New investigator Pilot Program in Bioinformatics, PACER Innovative Discovery Award, JDRF Innovative Immunotherapies Award,
Epitope Tag Conjugation System Provides Versatility for Multiplex Assay Design
Authors: Miller Amber D , Fails Danielle , Doyle Kristin , Hernandez Alyssa , Spencer Mike 
Published: August 2023

Abstract
With the ever-growing use of multiplex assays in research, therapeutics, and diagnostics, the need to conjugate antibodies to dyes, enzymes, and other materials is increasing. While there are many challenges to overcome regarding conjugation, the largest hurdle is maintaining antibody activity after conjugation. To combat these conjugation challenges, Alpha Thera developed a conjugation system that utilizes a small adapter protein to label antibodies quickly and site-specifically, resulting in antibodies that maintain their activity post conjugation. The adapter protein contains a customizable C-terminal region that can be modified to contain diverse labels including fluorescent dyes, click-chemistry, drugs, and epitope tags. Incorporating the epitope tag conjugation system with Fortis Life Sciences anti-epitope tag detection antibodies, we developed an easy and adaptable system for generating conjugated antibodies to use in multiplex assays.
We used this system to detect staining of multiple makers including CD3, TIM3, PCNA, and actin via flow cytometry, IHC, ICC, and western blot. One of the tested antibodies retained normal activity post-conjugation even though prior attempts with metal tagging via sulfhydryl groups resulted in lost activity. The vast array of conjugated anti-epitope tag antibodies allows for the selection of specific dyes/materials that are optimized to your desired assays. Additionally, since the secondary antibody is detecting the epitope tag, multiplexing is simplified, and antibody host is rendered irrelevant. This epitope tag system provides a simple, site-specific conjugation method that has incredible adaptability for any multiplex assay.
Robust Expansion Of Human Blood-derived γδ T Cells Using A Chemically-defined Medium
Authors: Zhang Chengkang , Roderick Karen , Burkall Amy , Kagan Elizabeth , Bossie Alexis 
Published: August 2023

Abstract
Gamma delta (γδ) T cells have recently emerged as an attractive candidate for cancer immunotherapy treatments due to their inherent cytotoxicity against cancer cells. Moreover, γδ T cells provide a platform for allogeneic cell therapies as they can recognize antigens independent of MHC recognition and do not cause graft-versus-host disease (GVHD). However, the lack of robust and GMP-compatible serum-free ex vivo expansion system for γδ T cells hinders the development of scalable expansion processes for advancing γδ T cell immunotherapies.
In the current study, we characterize the expansion of human peripheral blood-derived γδ T cells from various donors using TheraPEAK® T-VIVO™ Medium, a novel chemically-defined cell expansion medium which is free of undefined and animal-origin components. Peripheral blood mononuclear cells (PBMCs) stimulated with zoledronic acid resulted in more than 1,000 fold expansion of γδ T cells in only 14 days with TheraPEAK® T-VIVO™ Medium containing 100 IU/mL human recombinant IL-2, without the need for human serum supplementation. Similarly, PBMCs stimulated with agonist antibodies targeting Vδ1 or Vδ2 T cell receptors (TCR) induced robust expansion of Vδ1 or Vδ2 T cells, respectively, in TheraPEAK® T-VIVO™ Medium. Collectively, these data support the use of TheraPEAK® T-VIVO™ Medium to advance the development of γδ T cell manufacturing processes.
A new benchtop system for simple and versatile introduction of macromolecules into human lymphocytes and CD34+ cells by microfluidic squeezing
Authors: Chehal Manreet , Ouellet Eric , Sikora Hanson Jacquelyn L , Binson Elinor , Liao Tina , Chau Phillip , Lambertz Ulrike , Kuo Grace , Yu Jessie , Chen Kevin , Dadgar Maisam , Dalton Bob , Gilbert Jonathan B , Sharei Armon , Eaves Allen C , Louis Sharon A , Kokaji Andy I 
Published: August 2023

Abstract
Intracellular delivery of molecules is a key step in biological research that enables the development of new methods of cell and gene-based therapies. Traditional methods for intracellular delivery are limited in a) delivering a wide variety of cargos, b) efficient delivery to a broad range of cell types, and c) preventing unwanted genetic dysregulation. To address these challenges, we have developed the CellPore™ system, a benchtop device for gentle, yet efficient delivery of cargos to a range of primary cell types. The CellPore™ system is a microfluidic platform that creates transient disruptions in the plasma membrane via mechanical deformation by applying pressure, thereby allowing cargo entry to the cytosol.
We developed a simple and straightforward workflow to determine optimal delivery conditions. This workflow consists of cell isolation, cargo preparation for delivery across a range of system pressures, followed by viability and delivery efficiency measurements. Optimized parameters were subsequently applied to deliver eGFP and mCherry mRNA as well as B2M-targeting Cas9 ribonucleoproteins to human unactivated T and CD34+ cells. Importantly, edited cells were functionally active after manipulation by the CellPore™ system, and subsequent studies in T cells revealed minimal transcriptional perturbations compared to electroporated samples.
These results demonstrate the importance of having a comprehensive understanding of the impact a delivery modality can have in a biological system. The CellPore™ system enables efficient delivery of cargos to target cells, while safeguarding key functional attributes. This simple workflow allows the CellPore™ system to be easily integrated into cell therapy research protocols.
Investigation of problematic dye combinations to inform optimal panel design in spectral cytometry
Authors: Stalbaum Kimberly , McKitrick Tanya R , Nguyen Chau M , Ellis Samantha , Sternberg Luise , Thomas Seddon Y 
Published: August 2023

Abstract
The adoption of high complexity flow cytometry is increasing as spectral analyzers and sorters can support detection of 40+ parameters. While experimental design and panel building can be straightforward for small and mid-sized conventional and spectral panels, high parameter panel building requires special considerations. Reaching the full capability of any cytometer requires the user to build a complex panel with all the available detectors in use. While smaller panels can rely on techniques such as separation of fluorophores based on laser excitation and emission peaks and selection of more spectrally clean dyes, increasingly complex higher parameter panels often require the use of problematic fluorophore combinations. Fluorophore combinations can be categorized as problematic for several reasons, including but not limited to; high spectral similarity, overlapping peak channel detection, high cross-laser excitation, and relative brightness. We present data intended to help inform the selection of fluorophores when building a complex panel. To address this, we designed mini panels composed of problematic fluorophore combinations to investigate which properties of such combinations contributed most significantly to overall panel usability, as well as to make recommendations for which fluorophores can be used together successfully. Data collected from both conventional and spectral cytometers showed that some combinations fail to resolve target populations or cause insurmountable compensation or unmixing errors, while others can be used together effectively. Thus, strategic dye selection can improve panel resolution as well as increase the number of detectable parameters, serving to maximize cytometer potential.
Cell identity, count, and viability for critical quality attributes using the Cellaca PLX image cytometer
Authors: Nitta Carolina Franco , Parker Aiyana , Hem Sopaul , Pierce Mackenzie , Smith Timothy , Ratnani Surbhi , Dobrowolski Marek , Gundimeda Srinivas , Lin Bo , Kuksin Dmitry , Chan Leo 
Published: August 2023

Abstract
Cell and gene therapy is one of the fastest growing fields for cancer therapeutics that heavily relies on robust and consistent instrumentations and technologies to verify and validate the fit-for-purpose critical quality attributes (CQA). Some of the key parameters required for satisfying CMC (Chemistry Manufacturing and Controls) criteria standards for cellular therapeutic products are routinely performed using flow cytometry. In the past decade, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated the sensitivity required for surface marker detection. We demonstrate the capability of the Cellaca PLX image cytometry system for surface marker population analysis, GFP and RFP cell viability, and cell health in comparison to flow cytometry. For immunophenotyping, PBMCs were stained with Hoechst/CD3/CD4/CD8, and viability detection of GFP and RFP-containing cell lines was performed by staining with Hoechst/RubyDead. Additionally, apoptosis was induced in Jurkat cells with staurosporine and cells stained with Hoechst/Caspase 3-488/RubyDead. All assays were analyzed on the Cellaca PLX and compared directly to the CytoFLEX. Results show similar surface marker populations: CD3 (76% and 81%, PLX and CytoFLEX, respectively), CD4 (43% and 42%), and CD8 (15% and 17%). Viabilities of RFP and GFP cells were equivalent on both platforms, and population percentages of Hoechst/Caspase 3/RubyDead positive cells were within 5% of each other. Our experiments demonstrate that the Cellaca PLX image cytometer can be of significant value to the Cell and Gene Therapy communities to satisfy several CMC criteria including high-throughput, high sensitivity, and low maintenance.
A rapid and high-throughput T cell immunophenotyping assay for cellular therapy bioprocess using the Cellaca PLX image cytometer
Authors: Nitta Carolina Franco , Pierce Mackenzie , Elia Jeanne , Ruiz Jen , Hipol Art-Danniel , Fong Nicolas , Qazi Henry , Kessel Sarah , Kuksin Dmitry , Mejia Eunice , Lin Bo , Smith Timothy , Croteau Josh , Schrantz Nicolas , Yang Xifeng , Chan Leo Li-Ying 
Published: August 2023

Abstract
Cellular therapies such as chimeric antigen receptor (CAR) T or NK cells undergo phenotypic characterization during discovery and development of novel therapies, biomanufacturing, and upstream patient cell processing for clinical use. Samples are routinely analyzed via flow cytometry for T cell, NK cell, B cell, and monocyte surface marker expression. Image cytometry systems perform cell-based assays that can be used as a convenient alternative to flow cytometry. Recently, a new high-throughput image cytometer, the Cellaca PLX system (Nexcelom, Lawrence, MA) was developed for immunophenotyping, transfection/transduction efficiency, and cell health assays. This new instrument can assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization that focuses on cellular therapeutic products. Here we demonstrate a rapid and high-throughput immunophenotyping and viability detection in peripheral blood mononuclear cells (PBMCs) using the Cellaca PLX. PBMCs underwent red blood cell (RBC) lysis and CD3 enrichment, were stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead surface marker kits and measured on the Cellaca PLX and three different flow cytometers for comparison. Results showed highly comparable cell populations between the flow cytometers and the Cellaca PLX suggesting that the image cytometer may provide a rapid and convenient alternative method for immunophenotyping. The proposed method can streamline workflows from sample staining to data analysis, quickly moving precious patient samples into downstream processes.
Establishment of a vaginal organoid system to study local resident memory T cell differentiation
Authors: Ulibarri Max R , Hasan Mohammad H , Mithila Farha J , Han Geongoo , Pacia Jessica , Gopinath Smita , Milner Justin , Beura Lalit 
Published: August 2023

Abstract
Over a million sexually transmitted diseases (STDs) are acquired every day worldwide, many of which preferentially target the female reproductive tract (FRT). Resident memory T cells (TRM) in the vaginal mucosa have proven protective roles against these STDs. Accordingly, a crucial objective of STD vaccinations is to establish a higher quantity and quality of TRM in the FRT. The vaginal epithelium has been implicated in regulation of local TRM differentiation and function, but the biological nature of this interaction has not been investigated. Here, we established a murine three dimensional (3D) vaginal organoid-CD8 coculture system to examine epithelial-T cell interactions at the molecular level. Differentiation of single epithelial stem cells led to the formation of 3D epithelial organoids. These organoids can be maintained long-term through serial passage and structurally resembled vaginal epithelium with a multilayer specialized epithelium. After culturing activated CD8 T cells with the organoids, we assessed the transcriptional and phenotypic composition of the T cells. We found that cocultured CD8 T cells were integrated into the extracellular matrix and acquired phenotypic and functional traits of in vivo TRM. This TRM-biased differentiation could be suppressed by a small molecule inhibitor of the cytokine TGF-β. Overall, the vaginal organoid coculture model recapitulates the in vivo environment far better than traditional 2D cell culture, while retaining high-throughput abilities. Importantly, it will enable highly reductionist experiments to examine the complex epithelial cell-T cell crosstalk to devise targeted approaches for improving CD8 T cell mediated immunosurveillance in the FRT.
Supported by grants from NIH (2P20GM109035 & P20GM121298) and by the Searle Scholars award.