bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying spatial immuno-oncological parameters of mouse tumors using Imaging Mass Cytometry
Authors: Pfister Thomas D , Raza Qanber , Lim Liang , Loh Christina
Published: August 2023

Identification of immuno-oncological (IO) processes that dictate tumor growth, metastasis, and immune response is essential for selecting promising drug candidates in preclinical models. Imaging Mass Cytometry™ (IMC™) is a proven high-plex imaging technology that enables deep characterization of the complexity of tumor tissue without disrupting spatial context while eliminating artifacts caused by tissue autofluorescence. Here, we showcase the Maxpar® OnDemand™ Mouse Immuno-Oncology IMC Panel Kit for application on mouse tumor tissues.

We compiled a 33-plex antibody panel to quantitatively assess IO-related processes and applied it to a tissue microarray containing a large variety of mouse tumors. We digitized high-plex data from mouse tissues using the Hyperion™ Imaging System and generated images demonstrating the detailed layout of the tumor microenvironment (TME). We further conducted single-cell analysis to identify specific populations of tumor and immune cells in the TME.

The Mouse IO IMC Panel Kit successfully identified pathophysiological processes such as immune cell infiltration and activation, signaling pathway activation, biomarkers of epithelial-to-mesenchymal transition (EMT), metabolic activity, growth, and the TME tissue architecture. Single-cell analysis of several highly relevant tumor types separated distinct cellular clusters representing tumor, immune, stromal, and vascular cells.

Our quantitative analysis of tumor composition revealed critical insights regarding prognostic parameters such as metastatic and growth potential of tumor cells and activity of immune cell infiltrates.

For Research Use Only. Not for use in diagnostic procedures.
Decoding the structural and cellular composition of the tumor microenvironment in multiple human cancers using Imaging Mass Cytometry
Authors: Pfister Thomas D , Lim Liang , Ouladan Shaida , Zabinyakov Nick , Raza Qanber , Loh Christina
Published: August 2023

High-plex imaging techniques such as Imaging Mass Cytometry™ (IMC™) have become key tools in understanding and decoding the spatial complexity of the tumor microenvironment (TME). The TME contains tumor-infiltrating lymphocytes that have been associated with positive therapeutic outcomes. IMC enables detailed assessment of cell phenotype and function using 40-plus markers simultaneously at subcellular resolution on a single slide without spectral overlap or background autofluorescence.

We customized the Maxpar® Human Immuno-Oncology IMC Panel Kit using antibodies from the Standard BioTools™ catalog to create panels for tissue-based immuno-oncology research. Data acquisition was performed using a Hyperion™ Imaging System. Cell segmentation was facilitated using an IMC Cell Segmentation Kit. A pixel classification approach and CellProfiler™ were applied for single-cell segmentation. HistoCAT™ was used for single-cell analysis to visualize protein expression in various cancer types via PhenoGraph clustering and t-SNE maps.

Our custom panels were applied to normal and cancer human tissue microarrays to phenotype and analyze cell populations in these tissues. We classified the activation state of immune cells, epithelial-to-mesenchymal transition (EMT) progression, and composition of the extracellular matrix. In-depth single-cell analysis quantitatively evaluated the cellular makeup and immune cell component in the TME of cancer tissues. This work demonstrates the capability of IMC to identify subcellular localization of cellular and structural markers, including quantitative and spatial identification of multiple immune parameters in the TME, in tumor microarrays of cancer subject samples.
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

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

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

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

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

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

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

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

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

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

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

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

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.
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

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

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

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 <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> 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 (<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

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

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

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 < 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

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

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

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

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

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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