bluuebunny/crossref_metadata_2025 huggingface dataset:
Restoration of CD4+ T helper cells without modulating the hepatic inflammatory pattern is not sufficient to prevent HCC
Authors: Manjili Masoud H , Isbell Madison , Koelsch Nicholas James , Mirshahi Faridoddin , Guo Chubquing , Idowu Michael , Austin Dana , Gelber Cohava , Wang Xiang-Yang Shawn , Sanyal Arun
Published: August 2023

Recent studies suggest that progression of nonalcoholic fatty liver disease (NAFLD) to hepatocellular carcinoma (HCC) is associated with a decreased frequency of the hepatic CD4+ T cells, as well as a predominance of the inflammatory immunological pattern. To determine whether the inhibition of chronic inflammation could prevent the progression of NAFLD to HCC, we used a novel LRP-1 agonistic peptide, SP16, during a progressive NAFLD in an animal model of NAFLD, i.e., DIAMOND mice. Tumor progression, circulating inflammatory cytokines as well as the hepatic immune cells were analyzed using 44-plex cytokine array and multi-color flow cytometry. Although SP16 restored the hepatic CD4+ T cells and slightly increased Th2 subset, it failed to modulate the CD4+ Th1 dominant pattern in the liver or prevent HCC. These data suggest that the hepatic immune pattern, which could produce a collective function independent from its cellular components, is more important than CD4+ T cell recovery during disease progression. Our data also suggest that administration of SP16 very early during NAFLD could shift a predominant Th1 pattern towards an equilibrium Th1=Th2=Th17 pattern which has been found to protect DIAMOND mice from from the progression of NAFLD to HCC. Future studies will determine if modulation of the inflammatory immune response by SP16 when administered early during NAFLD progression will prevent HCC.

This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Breast Cancer Research Program under Award No. W81XWH2210793, Massey Cancer Center Multi-Investigator Award, grant number 2017-MIP-02, NIH R01DK105961, and VA Merit Award 1I01BX003275. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Department of Defense. Services and products in support of the research project were generated by the Virginia Commonwealth University Flow Cytometry Shared Resource, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059.
High-throughput method to analyze the cytotoxicity of CAR T Cells in a 3D tumor spheroid model using image cytometry
Authors: Patel Samir , Zurowski David , Hui Daniel , Ka Mignane , Hernandez Charles , Love Andrea , Lin Bo , Moore Andrea , Chan Leo Li-Ying
Published: August 2023

Chimeric antigen receptor (CAR)-T cell therapy is an antigen-dependent cellular therapy that has gained considerable traction in the field of cancer immunotherapy. Currently, there are six FDA-approved CAR-T cell therapies, which all target the CD19 or BCMA antigens for hematologic B cell malignancies. In recent years, a strong focus has been placed on CAR T cell therapy discovery for solid tumors, which may better recapitulate physiological conditions, thereby potentially improving the selection of CAR construct candidates. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to better assess CAR-T cell therapies against solid tumors. Recently, plate-based image cytometry has emerged as a method to investigate and characterize CAR T cell functions in a high-throughput manner. Image cytometry has demonstrated capabilities in analyzing transduction efficiency, cell proliferation, and cytotoxicity for CAR T cell therapy. With the development of 3D spheroid models, image cytometry may provide the necessary tools and applications for CAR T cell therapy discovery geared towards solid tumors. In this work, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. Herein, we demonstrate the use of high-throughput image cytometry to characterize PSMA CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids and simultaneously monitor location of the T cells in vitro. The proposed method can effectively assess cell-mediated 3D tumor spheroid cytotoxicity and generate time- and dose-dependent results.
Identifying next generation Ig selective cleaving enzymes for treatment of autoimmune diseases using IMPACT platform
Authors: Mande Purvi , Hoyt Emily , Shin June , Pellerin Alex , Le Nam , Bengeri Soumya , Anderson Jordan , Hoel Chris , Higginson-Scott Nathan , Otipoby Kevin , Mascanfroni Ivan
Published: August 2023

Dysregulated humoral immune mechanisms result in immunoglobulin production that is pathogenic and contributes to a wide range of autoimmune diseases and transplant rejection. Proteases derived from human pathogens can specifically cleave these immunoglobulins (Ig) and can potentially eliminate circulating, cell-surface and immune-complex Ig classes and modulate Ig-mediated autoimmunity and inflammation, providing a novel opportunity to treat antibody-mediated diseases.

We describe here the discovery, generation, and functional characterization of Ig specific proteases. These proteases were identified using Seismic Therapeutic’s IMPACT platform and tested for their ability to cleave intact Ig subclasses and Ig isotypes in a high-throughput CE-SDS cleavage assay. Additionally, we found that these enzymes cleave soluble and cell bound Ig in human blood, dramatically reducing effector function.

We believe that these subclass and isotype specific cleaving proteases will provide an opportunity to develop novel targeted therapies for various autoimmune diseases where autoantibodies are the key drivers of the disease. In addition, we believe that they will be potentially better tolerated compared to current therapeutic interventions such as plasma exchange and other immunosuppressive therapies.
The role of M1 to M2 macrophage polarization in the etiology of idiopathic gastroparesis: GWAS perspective
Authors: Smieszek Sandra P
Published: August 2023

Gastroparesis is a serious medical condition characterized by delayed gastric emptying. An innate immune dysregulation and injury to the interstitial cells of Cajal and other components of the enteric nervous system is likely central to the pathogenesis of gastroparesis. We have done a GWAS comparing idiopathic and diabetic cases with controls as well as contrasting idiopathic versus diabetic cases.

We report a novel genetic association of SLC15A4 locus with idiopathic gastroparesis. This signal is driven by multiple variants with top missense variant SLC15A4:NM145648:exon2:c.T716C:p.V239A, rs33990080. Altogether, among EUR cohort we report 69 carriers out of 214 versus controls 165 out of 896 (MAF: cases 0.18; MAF: controls 0.09) and OR: 1.9 (p-value logistic model 10^−6). The significant effect persists when idiopathic cases are compared with diabetic cases as the MAF of diabetic cases (0.10) is comparable to that of population controls (0.09).

Solute carrier family 15 (SLC15) A4 is a lysosome-resident amino acid/oligopeptide transporter that is preferentially expressed in immune cells. It is required for TLR7/9-dependent type I interferon production and plays a critical role in the pathogenesis of lupus and colitis. SLC15A4 was shown to mediate M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress. Previous reports have shown that when the anti-inflammatory M2 macrophages are switched to pro-inflammatory M1 macrophages, delayed GE was evident in animal models. Current genetic findings suggest that a mechanism directly connected to macrophage polarization may be implicated in a subset of idiopathic gastroparesis patients.
Follicular B cell derived CD21lo B cells are immediate precursors to autoreactive extrafollicular antibody secreting cells
Authors: Zhu Danni , Castrillon Carlos , Carroll Michael C
Published: August 2023

Systemic lupus erythematosus (SLE) is an incurable autoimmune B cell disorder that is in part due to ineffective clearance of nuclear antigens and activation of the TLR pathway. The extrafollicular (EF) differentiation pathway of antibody secreting cells (ASCs) is recognized to play a prominent role in generating pathogenic antibodies in both mice and human. For example, a novel population of CD21lo CD11c+ B cells identified in both mice (age-associated B cells) and human (DN2 cells) are thought to be a major source of autoreactive EF ASCs. However, the developmental kinetics and cellular origin of EF ASCs remain elusive. To track early events of B cell break of tolerance and EF ASC generation, an adoptive transfer system was established, where WT B cells are transferred into the autoreactive BCR transgenic 564Igi host enriched in nuclear antigens. By introducing competing populations of WT and TLR7 deficient B cells, we demonstrated that the differentiation of EF ASCs requires TLR7. To examine the kinetics of autoreactive B cell proliferation and EF ASC differentiation, a Cell Trace labeling approach was used. We found that donor B cells require at least 7 divisions to differentiate to EF ASCs and that TLR7 deficient B cells are gradually outcompeted by WT at each division. Interestingly, CD21lo CD23neg B cells were highly proliferative, expressed CD11c, and were sensitive to TLR7 defect. Our interpretation is that they are likely the immediate developmental precursors of EF ASCs. These findings advance our current understanding of the fundamental biological circuitry behind EF-derived autoantibody producing cells and potentially point to future avenues for therapeutic development.

Supported by NIH (5R01AR074105-05), CIHR Doctoral Foreign Study Award (DFSA) (DFD-181607)
Impact of B cell specific neutral lipid accumulation on B cell activation and function
Authors: Song Wenxia , Halpern Clarissa , Hu Cecilia , Lazzaro Michelle , Shapiro Melanie , Sherman Emily , Fan Sherry , Tyrlik Adam , Seeley-Fallen Margaret Kathryn
Published: August 2023

Obesity and its resultant inflammation have been shown to lead to a multitude of disease states, including Type II diabetes and cancer. Immune cells play a role in perpetuating obesity related diseases. B cells have been shown to infiltrate the visceral adipose tissue of obese individuals, where they activate other immune cells and produce autoantibodies. This study examined the impact of B cell neutral lipid accumulation on B cell function and activation, using B cell specific comparative gene identification-58 (CGI-58) knockout mice (bCKO). CGI-58 is the co-activator of adipose triglyceride lipase, a key enzyme initiating lipid droplet lipolysis. CGI-58 knockout and flox control mice were fed a low-fat diet for 16 or 32 weeks. B cells from bCKO mice exhibited a higher level of neutral lipid accumulation than other cell types and flox control B cells. Unimmunized bCKO mice showed increased percentages of spontaneous germinal center B cells compared to flox controls. Isolated bCKO B cells survived in culture significantly better than flox control B cells, which was concurrent with decreased levels of cellular and mitochondrial ROS and mitochondrial membrane potential in bCKO B cells compared to flox B cells. Furthermore, bCKO mice had increased numbers of crown-like structures in their visceral adipose tissues and elevated protein levels in their urine, predictors of metabolic disorder. Together, our results suggest that neutral lipid accumulation in B cells alone leads to non-specific survival and activation of B cells by downregulating ROS production and modulating mitochondrial functions.

Supported by a grant from NIH (RO1 GM064625)
Multiomic Spatial Phenotyping of the Tumor Immune Microenvironment at Single Cell Resolution
Authors: Jhaveri Niyati , Ji HaYeun , Dikshit Anushka , Yuan Jessica , Doolittle Emerald , Zhou Steve , Srinivasan Maithreyan , Darling Julia Kennedy , Braubach Oliver
Published: August 2023

The Tumor microenvironment (TME) is critical in cancer development, progression, and control. Immunological components within tumors, known as the tumor immune microenvironment (TIME), have been implicated in cancer progression. Effective strategies for cancer immunotherapies will require a deep understanding of factors that shape the TME and TIME. Here, we describe a spatial multiomics assay utilizing RNAscope™ ISH technology paired with high-plex whole-slide spatial phenotyping with the PhenoCycler®-Fusion platform. This two-step approach is compatible with human FFPE tissues and enables characterization of the TIME more accurately by detecting RNA and protein markers on serial sections.

We performed ultrahigh-plex spatial phenotyping on FFPE tumor tissue sections, using an antibody panel designed for immune cell phenotyping, evaluation of immune contexture and proliferation across the TME. On a serial section, we ran the RNAscope HiPlex v2 assay using the PhenoCycler-Fusion system. This assay consisted of a 12-plex immuno-oncology panel of RNA target probes, which detect macrophages, chemokines, and cytokines.

In this proof-of-concept study, we demonstrate multiomic spatial phenotyping on the PhenoCycler-Fusion platform. Analysis of the resulting multiplex imaging data revealed structural organization of cells within the TME and activation states of immune cells. Together, this information provides a more complete functional map of immune cells within the TME and TIME and thereby enriches our understanding of tumor biology that may be deterministic of immunotherapy responsiveness.
Optimization of CRISPR-Cas9 Mediated Knockout Protocol in Primary Human Myeloid Cells
Authors: Ruvalcaba Angel , Reijneveld Josephine F , Suliman Sara
Published: August 2023

Myeloid cells are an important player in the immune system, and form the first line of defense against pathogens, such as Mycobacterium tuberculosis (Mtb), the causative pathogen for tuberculosis (TB) disease. They include macrophages, a target of Mtb infection, and dendritic cells (DCs), which present Mtb-derived antigens to prime and activate T cells. In order to investigate myeloid-specific host factors involved in the response to Mtb infection, we sought to optimize a protocol to genetically edit primary human monocyte-derived DCs and macrophages for subsequent infection experiments with Mtb. We built on prior published protocol by Hiatt, et al. to edit monocyte-derived DCs and macrophages using nucleofection with ribonucleoproteins (RNP), which are Cas9 recombinant protein complexed with multiplexed guide RNA (gRNA) molecules. We tested reaction volume, RNP concentration, rest time, and nucleofection programs. We found that a molar ratio of Cas9:gRNA was most efficient at 1:2 at 1.8μM final RNP concentration in combination with a 2hr rest time after nucleofection before adding differentiation supplement. Lonza nucleofection program EA100 proved to be the optimal program with the most recovered cells. In conclusion, we optimized a robust protocol for editing and culturing edited primary monocyte-derived DCs and macrophages. This opens opportunities for interrogation of candidate TB risk genes such as Fumarylacetoacetate hydrolase (FAH). These cells can now be used for mechanistic studies of Mtb infection to define mechanisms of pathogenesis.
FastqToGenCounts: Converting Fastq Files to Gene Counts Matrices
Authors: Loecker Josh , Bessell Brandt
Published: August 2023

FastqToGeneCounts is a computational pipeline built on Snakemake to process and analyze bulk RNA sequencing data to determine gene expression. It can handle raw data from the Gene Expression Omnibus or local FastQ files and runs on a supercomputing cluster. The primary function of FastqToGeneCounts is to align FastQ files to a reference genome. To ensure accurate outputs, it also offers trimming, quality control, and contaminant screening options.

One of the benefits of using FastqToGeneCounts is that it addresses the issues commonly encountered with traditional alignment workflows. These include the need for manual file naming and directory setup, which can lead to errors and problems resuming failed workflows. Additionally, these workflows often require many resources requested at once, leading to long “wait times” for cluster access. Modifying parameters can also be challenging, as most settings are not localized to a single file.

In contrast, FastqToGeneCounts integrates with Snakemake to improve resume-ability, minimize resource usage, and provide easy parameter modification. Jobs can be resumed from a failed state, only the minimum required resources are requested to reduce waiting time, and a single YAML file can be used to configure parameters within the pipeline.

FastqToGeneCounts has been used to analyze four immature natural killer datasets and has successfully determined gene expression in these samples. It is capable of analyzing a single raw RNA-seq input file in approximately 15 compute minutes, and additional files do not increase runtime due to Snakemake’s high interoperability with SLURM. FastqToGeneCounts removes poor-quality reads from downstream analysis and presents results in an easy-to-read report.
Artificial Intelligence (AI) based approach for identifying biomarkers associated with neutralizing antidrug antibodies to Factor VIII used in Hemophilia A treatment
Authors: Rawal Atul , Kidchob Christopher , Ou Jiayi , Yogurtcu Osman N , Yang Hong , Sauna Zuben E
Published: August 2023

Lack of clinically validated markers associated with the development of neutralizing antibodies (inhibitors) to the replacement protein-drug, Factor VIII (FVIII) is a major challenge in managing Hemophilia A (HA). By utilizing Machine Learning (ML) and Explainable AI (XAI) we identified and ranked relevant variables (biomarkers) that could be predictive for developing inhibitors to FVIII drugs in hemophilia A patients. The dataset used for the study was derived from the My Life Our Future (MLOF) repository and included variables such as age, race, ethnicity, mutations in the F8 gene and the Human Leucocyte Antigen Class II (HLA-II) type. We computed the number of foreign FVIII derived peptides, based on the alignment of the endogenous FVIII and infused drug sequences, and the foreign-peptide HLA-II molecule binding affinity (using the NetMHCIIpan algorithm). The complete dataset with the added variables was trained and validated using multiple ML classification models to identify the top performing model, which was chosen for further processing with XAI via SHAP (SHapley Additive exPlanations) to identify the variables critical for the prediction of FVIII inhibitor development in a hemophilia A patients. The top five variables for predicting inhibitor development based on SHAP values are: (i) the baseline activity of the FVIII protein, (ii) mean affinity of all foreign peptides for HLA DRB 3, 4, & 5 alleles, (iii) mean affinity of all foreign peptides for HLA DRB1 alleles, (iv) the maximum affinity among all foreign peptides for HLA DRB1 alleles, and (v) F8 mutation type.

This research was supported by funds from the Hemostasis Branch/Division of Plasma Protein 344 Therapeutics/Office of Tissues and Advanced Therapies/Center for Biologics Evaluation and Research 345 of the U.S. Food and Drug Administration and in part by an appointment to the Research Participation 346 Program at the Center for Biologics Evaluation and Research administered by the Oak Ridge Institute 347 for Science and Education through an interagency agreement between the U.S. Department of Energy 348 and the U.S. Food and Drug Administration. The MLOF program was developed as a partnership 349 between NHF, ATHN, Bloodworks Northwest, and Bioverativ and supported financially by 350 Bioverativ, NHF, Bloodworks Northwest, and ATHN.
Expanding panel size in spectral flow cytometry with StarBright Dyes excitable by the ultraviolet (355 nm), violet (405 nm), blue (488 nm) and yellow (561 nm) lasers
Authors: Blundell Michael , Sanderson Sharon
Published: August 2023

The recently launched fluorescent dyes from Bio-Rad, StarBright Dyes, deliver tunable brightness and spectral properties, greater stability, improved lot-to-lot reproducibility, and spectral consistency. Specifically designed for multicolor flow cytometry with researchers needs in mind, StarBright Dyes address the common pain points in flow such as brightness, broad emission spectra, staining consistency, and ease-of-use, solving issues of signal resolution when constructing complex panels. These bright dyes have the additional benefit of a unique full spectral profile, making them ideal for inclusion in spectral flow cytometry, when creating multicolor panels.

Here we present data of our newest additions to the StarBright range, the StarBright Blue and StarBright Yellow Dyes on a five-laser spectral cell analyzer. Both dye series are bright and allow expansion across the 488 and 561 lasers giving more dye choice for all panel sizes but are particularly useful when large panel design is required.

In this study we show, when StarBright Blue and StarBright Yellow Dyes are combined with other members of the StarBright Dye range, polymer dyes and traditional fluorescent dyes, very large immunophenotyping panels can be constructed allowing identification of many peripheral blood subsets. In addition, novel dye combinations can be identified increasing the numbers of dyes and therefore markers identified from one sample.

When combined with their ability to be fixed in both PFA based and alcohol-based fixatives, with minimal spectral changes, and to be pre-mixed for up to 33 days, the flexibility of StarBright Dyes means they are ideal for new and expanding existing panels regardless of the type of flow cytometer being used.
Mapping the gene space at single-cell resolution with gene signal pattern analysis
Authors: Venkat Aarthi , Damo Martina , Joshi Nikhil S , Krishnaswamy Smita
Published: August 2023

In single-cell RNA sequencing analysis, several computational methods have been developed to map the cellular state space, but little has been done to map the gene space. A mapping that preserves gene-gene relationships within the dataset is particularly useful for characterizing cellular heterogeneity within cell types, where boundaries between cell subpopulations are often unclear or even arbitrary.

Here, we present gene signal pattern analysis, a new paradigm for analyzing single cells. We build a cell-cell graph and design a dictionary of diffusion wavelets, capturing a multiscale view of the cell space. We then transform genes by the dictionary and learn a reduced gene representation. Given the gap in prior research for this problem, we design nine alternative strategies and three benchmarks for evaluating preservation of gene-gene relationships, all of which are outperformed by diffusion wavelet-transformed signals. We also define, calculate, and evaluate localization, a key property of a gene signal on the cellular graph.

We demonstrate the utility of gene signal pattern analysis on T cells from a mouse model of peripheral tolerance in skin. The gene space mapping reveals a continuum of gene signals characterized by T cell subtypes and transcriptional programs related to effector function and proliferation. Furthermore, we built a multiscale manifold of 48 melanoma patient samples, demonstrating the ability of our method to characterize differences between responders and non-responders to checkpoint immunotherapy. Together, we show gene signal pattern analysis, through methodology from graph signal processing, spectral graph theory, and machine learning, represents an avenue for future research in scRNA-seq analysis.

Gruber Foundation
Flexible and high-throughput approach for capturing large number of single cells simultaneously using microwell-based technology
Authors: Nguyen Rosary , Hlathu Zorine , Gordon Chelsea , Zhao Xueying , Moskwa Jamie , Jensen Devon , Acob Ricelle A , Wang Larry , Ayer Aruna
Published: August 2023

Single-cell RNA-seq is a powerful method for studying complex cell populations. As opportunities grow for single-cell multiomics methods in translational research, there is an increased need for high-throughput approaches capable of efficiently capturing and analyzing large numbers of cells simultaneously. Current assays on scRNA-seq platforms have limited throughput due to the low cell recovery and high multiplet rates with high cell inputs.

Here we demonstrate the feasibility of profiling the single-cell gene expression of hundreds of thousands of cells in a single experiment using an eight-lane microwell based cartridge. We also increased the cell label diversity of cell capture beads to enable high cell loading with a lower rate of barcode collisions. To show the range of cell capture capabilities of the eight-lane microwell cartridge, we loaded different numbers of cells per lane ranging from 100 to over 40,000 cells. To evaluate the multiplet rates and sensitivity, we labeled two different cell types with antibody-based sample multiplexing prior to cell capture. Cell multiplet rates were also assessed through Sample Tag analysis. Detection of Sample Tags used showed no contamination of Sample Tag between samples. In addition, less than 0.5% barcode collision rate was detected from 360,000 viable cells captured with a bead. Targeted mRNA profiling had high correlation (R2 > 0.95) of gene expression across cell capture lanes. This study demonstrates that the eight-lane microwell-based cartridge technology provides flexibility and high-throughput cell capture of thousands of cells and could be readily extended to other existing single-cell technologies such as surface protein marker detection and immune profiling.
Universal, open MHC-I for rapid ligand loading and enhanced complex stability across classical and non-classical allotypes
Authors: Young Michael C , Sun Yi , Woodward Claire H , Danon Julia N , Truong Hau V , Sgourakis Nikolaos G
Published: August 2023

The instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents an important biochemical challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs) for autologous therapy development. Here, we leverage the coupled binding between ligand and the light chain (beta-2 microglobulin, β2 m) to the MHC-I heavy chain with the aim of developing conformationally stable molecules by engineering a disulfide bond to bridge the interface between the heavy and light chain. The resulting MHC-I molecules are stabilized in a ligand-receptive/open state, exhibit favorable ligand exchange properties, and are readily loaded with moderate affinity ligands to afford properly conformed protein complexes of enhanced stability compared to the wild type. We demonstrate the generality of the structural design using polymorphic HLA representatives covering multiple HLA-A and B supertypes, the nonclassical and oligomorphic HLA-Ib molecules, as well as MHC-like molecules, MR1 and CD1. Our platform allows spontaneous and rapid ligand exchange, which enables a range of approaches for ligand screening and the functional screening and characterization of polyclonal TCR repertoires.

Supported by grants from NIH (R01AI143997, R35GM125034, U01DK112217).
Comparison of CITE-Seq assay noise between leading microwell and droplet systems
Authors: Campbell Tracy L , Martin Jody , Song Hye-Won , Wang Larry , Ayer Aruna
Published: August 2023

CITE-Seq experiments in combination with whole transcriptome analysis (WTA) can provide researchers with invaluable multiomics data to answer critical cellular biology questions. In selecting the right platform to answer these questions, researchers are confronted with a myriad of options and the benefits of each may not always be clear when comparing summary metrics alone.

Proteomics assays using oligo-conjugated antibodies have metrics beyond sensitivity to consider when choosing the right platform, for example antibody specificity as well as antibody oligo-mediated noise. Noise from negative populations in a CITE-Seq experiment can add to overall experiment costs while complicating downstream analysis and obscuring conclusions.

In this study, we are comparing a CITE-Seq assay in combination with WTA between the leading microwell and droplet-based systems while controlling for final antibody staining concentrations.

We demonstrate that the CITE-Seq assay on the microwell-based system has higher resolution and less noise than the droplet system when comparing across two PBMC donors.

© 2023 BD. All rights reserved. NPM-0171 (v1.0) 0123
Fast Preparation of Peptide Tools to address new emerging Viruses: Case example SARS-CoV-2
Authors: Castro Aaron , Pfeil S , Holenya Pavlo , Reimer Ulf , Knaute Tobias , Zerweck Jonny , Eckey Maren , Kern Florian , Drosch Michael , Alem Aude Marie , Wenschuh Holger , Schnatbaum Karsten
Published: August 2023

The emergence of new virus species or variants poses a major challenge for healthcare systems. Countermeasures, including vaccine development, need to be developed without delay to ensure success. Vaccine and antibody development depend on highly efficient tools for antigen discovery, epitope mapping, diagnostics and (clinical) immune monitoring.

Here we present a workflow for peptide tool development that can be rapidly adapted to emerging viruses. It combines bioinformatics algorithms and an ultra-high throughput peptide synthesis method alongside high-content assay formats.

Data from different SARS-CoV-2 studies are presented addressing elucidation of both cellular and humoral immune responses. Robust spike-specific cellular immune responses were measured using SARS-CoV-2 Spike PepMix™ peptide pools during infection and/or vaccine response monitoring [1]. Using epitope mapping (matrix) pools an immunodominant T-cell epitope was identified that may contribute to the optimization of existing vaccines [2]. In addition, the immune response of Covid-19 convalescents against all major SARS-CoV-2 proteins was analyzed [2]. Humoral immune response analysis by high-density peptide microarrays identified immunodominant B cell epitopes as starting points for the development of alternative vaccines and diagnostic tests [3].

In conclusion, we show that readily available and tailored peptide formats, as exemplified for SARS-CoV-2, are important tools that contribute to the fast development and optimization of vaccines and diagnostic tests for emerging viruses of concern.
Harnessing violet and ultraviolet fluorophores for flexibility in T cell panel design
Authors: Meng Lihao , Sternberg Luise , Ruiz Natalie , Funatake Castle J
Published: August 2023

The cytotoxic capabilities of T cells are being leveraged for immunotherapeutic approaches in the fight against cancer. These treatments have demonstrated significant promise thus far and warrant refinement to increase efficacy, reduce unwanted effects, and expand their capabilities to more diverse tumor types. As such, continuing to broaden our understanding of T cell biology at a basic research level is critical for developing the next generation of T cell immunotherapeutics.

Spectral flow cytometry enables recognition of the unique spectral signature of individual fluorophores and facilitates the addition of more antibodies by accommodating fluorophore combinations which are incompatible on conventional cytometers. An increased availability of fluorophore-conjugated antibodies for surface and intracellular antigens in the ultraviolet and violet laser range enable researchers to deepen their understanding of T cell activation and function with more flexibility in adding markers of interest. Here, we developed an optimized panel for the investigation of human T cell differentiation, activation, proliferation, and function. This panel demonstrates consistent and robust characterization of in vitro-activated human T cells, while leaving most channels on the blue, yellow-green, and red lasers available for additional markers of interest. This approach lends itself additional customization, facilitating deeper insights from a single, limited sample enabled by the broad availability of fluorophore-conjugated antibodies supporting these laser lines.

Thermo fisher Scientific
Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge
Authors: Cusimano Gina , Gary Ebony N , Tursi Nicholas J , Warner Bryce M , Connors Jennifer , Parzych Elizabeth M , Griffin Bryan D , Bell Matthew , Ali Ali R , Frase Drew , Hojecki Casey , Canziani Gabriela , Chaiken Irwin , Kannan Toshitha , Moffat Estella , Embury-Hyatt Carissa , Wootton Sarah , Kossenkov Andrew , Patel Ami , Kobasa Darwyn , Kutzler Michele A , Haddad Elias , Weiner David B
Published: August 2023

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity and mortality. Furthermore, the aged patient population has suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mouse models. Aged mice had altered cellular responses, including decreased IFNγ secretion and increased TNFα secretion as compared to young mice. Aged mice had significantly decreased binding and neutralizing antibodies in their serum compared to their young counterparts. Co-immunization with the plasmid-encoded molecular adjuvant adenosine deaminase-1 (pADA) enhanced cellular responses and expanded the breadth and affinity of humoral responses in aged mice. pADA co-delivery also altered the gene expression profiles of lymph node lymphocytes in aged animals. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a strong TH1 gene profile and decreased FoxP3 gene expression. Upon challenge pADA co-immunization decreased viral loads and age-associated morbidity and mortality in mouse-adapted and ACE2 transgenic SARS-CoV-2 challenge models. These data support the use of aged mice as a model for age-associated decreases in vaccine immunogenicity and increased in infection-mediated morbidity and mortality in the context of SARS-CoV-2. These studies provide further support for the use of adenosine deaminase as a molecular adjuvant in the elderly.

This work was supported by NIH NCI award T32 CA09171 (ENG) and NIH NIAID award T32-AI-055400 (EMP) and The Wistar Institute coronavirus discovery fund with additional support from CEPI/Inovio Pharmaceuticals.
Culturally-specific education can change perception of risk of Human Papillomavirus infection and need for vaccination
Authors: Poole Brian D , Redd David S , Altman Jessica D , Jensen Jamie L , Sloan-Aagard Chantel D , Crook Triston B , Asay Aaron E , Nielson Bryce U , Bodily Ruth J , Miner Dashiell S
Published: August 2023

Safe and effective vaccines have been developed that protect against high-risk strains of HPV, but uptake is relatively low. We previously identified factors such as sexual attitudes and HPV knowledge that impact the intent of Christian parents to vaccinate their children against HPV. One of the major factors that decreased vaccine utilization was the belief that they did not need to vaccinate their children because of their moral beliefs. We hypothesized that culturally specific interventions in the form of short videos would be effective at improving HPV vaccine attitudes. We made three short educational videos, one with a Christian focus, one informational about HPV, and one control. Videos were distributed electronically with accompanying surveys, and attitudes were measured before and after watching a randomly selected video. The religious-focused and educational interventions significantly (P=0.001) improved attitudes about HPV vaccination. The religiously-focused video also significantly diminished the belief that the HPV vaccine is unnecessary because of a family’s values (p=0.023). Parents significantly credited both interventions with improving their intent to vaccinate their children against HPV (p<0.001 for both). These results suggest that culturally focused educational interventions are effective at influencing vaccine attitudes, even when those attitudes are based on religious or cultural feelings. Highly specific interventions are likely to be necessary for optimal improvement in vaccine hesitancy.

Supported in part by a research grant from Investigator-Initiated Studies Program of Merck Sharp & Dohme LLC.
Altered antibody profiles after receipt of third dose SARS-CoV-2 mRNA vaccines in solid organ transplant recipients
Authors: Lee Jenny MinChen , Shapiro Janna R , Lee John S , Sitaris Ioannis , Peralta Stephanie R , Li Maggie , Wouters Camille , Karaba Andrew H , Blankson Joel N , Tobian Aaron A R , Werbel William A , Pekosz Andrew , Klein Sabra L
Published: August 2023

Immunosuppressed patients, including solid organ transplant recipients (SOTRs), developed significantly lower antibody responses and had more breakthrough infections after two doses of COVID-19 mRNA vaccines, as compared with immunocompetent individuals. The aim of this study was to characterize vaccine-induced antibody responses, including IgG binding antibodies and live virus neutralizing antibodies (NAb) against the vaccine strain and Omicron variants, BA.1 and BA.5, as well as the levels of the IgG subclasses, IgG1-4, in SOTRs. Serum samples from SOTRs were analyzed at pre-vaccination, 30-days, and 90-days following the third dose COVID-19 mRNA vaccines (D3). For several measures, antibody responses were compared with healthy controls (HC). A third dose vaccination increased anti-spike (S) IgG titers in response to the SARS-CoV-2 vaccine and Omicron variants from pre- to post-D3 in SOTRs. Anti-S IgG responses to Omicron variants, however, were significantly lower than to the vaccine strain. SOTRs also had low live virus NAb titers to the vaccine and Omicron BA.1 variant, with many individuals having no detectable NAb. The subclass-specific anti-S IgG antibodies against the SARS-CoV-2 vaccine strain predominantly consisted of IgG1, IgG3, and IgG4 in SOTRs, which resembled the subclass profiles in HC at post-D3. Together, our findings illustrate that while a booster dose of mRNA vaccine induces antibody responses, including IgG1 and IgG3, which are the dominant antiviral IgG subclasses, in SOTRs, these responses are still lower than immunocompetent individuals. Therefore, consideration should be given to different formulations and doses of COVID-19 vaccines in people with severely compromised immune systems.
Boosting location of COVID-19 mRNA vaccine regulates the germinal center reaction
Authors: Jiang Wenxia , Yuan Xue , Renshaw Scott , Luo Wei
Published: August 2023

Germinal centers (GCs) are the specialized sites for B cells to differentiate into long-lived plasma cells or memory B cells during vaccinations. COVID-19 mRNA vaccines require boosting to mount high levels of neutralizing antibody responses. mRNA vaccines induce persistent GCs in the draining lymph nodes (dLNs), which might change the microenvironment for subsequent immunizations. It is unclear whether the site of booster immunization (ipsilateral versus contralateral) will affect the GC responses in the dLNs. To test this, we compared GC responses induced by ipsilateral and contralateral booster vaccination three weeks post the primary immunization in mice. Although these two boosting strategies induce similar numbers of GC B cells in the dLNs, we discovered that ipsilateral boosting elicits more SARS-CoV-2 RBD-specific GC B cells than contralateral boosting. However, very few RBD-specific GC B cells were differentiating into plasma cells as probed by BCL6low IRF4high PC precursors within the GC B cell population. Serum antibody levels were comparable between these boosting strategies, suggesting the memory recall response was not different. These studies suggest boosting location of COVID19 mRNA vaccines might affect RBD-specific GC B cell responses. Future studies are needed to investigate whether boosting location may impact the persistence of neutralizing antibody response induced by COVID19 mRNA vaccines.
Visualizing the early immune response to Mycobacterium tuberculosis infection in macaques immunized with intravenous BCG
Authors: Bucsan Allison N , Darrah Patricia A , Dahlvang James D , Lehman Chelsea C , Robertson Molly A , Kamenyeva Olena , Kabat Juraj , Fabozzi Giulia , March Kylie , Via Laura , Walker April , Roederer Mario , Seder Robert
Published: August 2023

Tuberculosis (TB) causes 1.6 million deaths annually and the standard human vaccine, intradermal (ID) Bacille-Calmette Guérin (BCG), does not prevent pulmonary TB. Previously, we showed that intravenous (IV) BCG immunization elicited robust TB-specific lung-resident T cell responses compared to ID BCG in rhesus macaques (RMs). Following Mycobacterium tuberculosis (Mtb) challenge, 6 out of 10 RMs had no detectable disease as demonstrated by bacterial burden, granuloma formation, and primary Mtb-specific immune responses. Together, these data suggest that Mtb is cleared rapidly from the lungs of IV BCG-vaccinated RMs. To investigate the vaccine-induced lung environment and anamnestic response that might mediate protection against Mtb early after challenge, we developed a high-dose Mtb infection model in RMs that allowed us to locate and live-image areas of Mtb infection in the lungs using confocal microscopy. RMs immunized for 5 months with either IV or ID BCG were challenged with Mtb Erdman mCherry and euthanized 4 or 11 days later to characterize the lung microenvironment. We observed reductions in bacterial burdens in the lungs of RMs that received IV BCG compared to ID BCG-vaccinated and unvaccinated controls by CFU plating, flow cytometry, and confocal microscopy despite using a high challenge dose. Increased T cells were quantified in the lungs of both IV and ID BCG-vaccinated RMs compared to unvaccinated controls by flow cytometry and confocal microscopy. This research provides an opportunity to mine immune correlates of protection in a preclinical model, at the site of infection, and at a post-challenge time point that has been minimally characterized.

NIH Intramural Program
Intramuscular mRNA prime plus intranasal adenoviral vector booster elicit robust respiratory mucosal IgA responses against SARS-CoV-2
Authors: Tang Jinyi , Wu Yue , Zeng Cong , Taylor Justin J , Zhu Guizhi , Weissman Drew , Liu Shan-Lu , Sun Jie
Published: August 2023

Current SARS-CoV-2 mRNA vaccines induce robust humoral and cellular immunity in the circulation, but its ability in eliciting respiratory mucosal immunity is less characterized. Here, we demonstrated that systemic mRNA expressing ancestral spike (mRNA-S) vaccination alone induced weak respiratory mucosal neutralizing antibody and cellular immunity, particularly against SARS-CoV-2 Omicron strain. In contrast, an immunization strategy combining systemic mRNA-S administration plus mucosal adenoviral vector expressing ancestral spike (Ad5-S) booster induced strong T cell, B cell, and antibody responses in the respiratory tract, which can last for a long time. Furthermore, we found that Ad5-S mucosal booster promoted robust IgA-producing B cells in the respiratory tract, which were correlated with the levels of mucosal S-specific IgA levels. Strikingly, these local IgA-producing B cells were more cross-reactive to the Delta and Omicron Spike proteins compared to those IgG-producing B cells. We further showed that CD4 T cell help was required for the development of IgA-producing B cell in the respiratory mucosa. Taken together, our study identified a vaccination strategy and its associated mechanisms for the induction of strong cross-reactive IgA responses, which were shown to correlate with optimal protection against breakthrough infection, especially by Omicron sub-lineages. Hence, our data provide insights into the rational design of next-generation SARS-CoV-2 mucosal vaccines required for the protection against infection by SARS-CoV-2 Omicron sub-lineages or future variants.
Mechanistic insight into immune amnesia and immunity induced by wild-type measles virus compared to live-attenuated measles vaccine
Authors: Chan Kwan Pui , Bartlett Maggie , Rubens Jessica , Brockhurst Jacqueline , Ghimire Shristi , Nguyen Benjamin H , Hauer Debbie , Griffin Diane E
Published: August 2023

Despite the availability of a safe and effective vaccine, measles still causes significant mortality and morbidity among children in developing countries. As a disease that induces immune amnesia with increased susceptibility to other infections but also life-long immunity to measles, little is known about the immunogenetics of B cells that elicit measles-specific antibodies (Abs) in natural infection vs vaccination. We therefore conducted single-cell RNA sequencing (scRNA-seq) of longitudinal peripheral blood mononuclear cells (PBMC) and lymph node (LN) samples from wild-type (WT) measles virus (MeV) and live-attenuated measles vaccine (LAMV)-infected rhesus macaques (RMs) and characterized their gene expression, B cell repertoires and clonotype changes via bioinformatic pipelines. Preliminary analysis of two RMs across four timepoints (one WT MeV- and one LAMV-infected RM) revealed similarities and differences in immune cell populations and B cell repertoires, providing important mechanistic insights into B cell clonal development in natural infection as compared to vaccination.

Supported by grants from NIH R01 AI153140 and the Jeffrey Richardson Fellowship
Serial intravascular staining reveals altered T cell trafficking in aged and SIV+ rhesus macaques
Authors: Potter E Lake , Brammer Daniel , Foulds Kathryn , Roederer Mario
Published: August 2023

Lymphocyte trafficking is required for lymphocyte development and maturation, immune surveillance and response, and for immunological memory. While trafficking is integral for a functional immune system, it may also play a role in immune dysfunction. Patients with HIV and the elderly have immune dysfunction characterized by increased inflammation, immune activation, and susceptibility to infections and cancer. As trafficking is implicated in these processes, we hypothesized that these cohorts may have altered trafficking kinetics, resulting in immunosenescence.

To determine the relationship between trafficking kinetics and immune dysfunction, we performed serial intravascular staining (SIVS) in healthy, aged, and SIV-infected rhesus macaques. SIVS uses sequential infusions of fluorescent antibody to label cells in vivo, enabling us to follow them as they migrate over time. In all animals, we found that naïve and central memory T cells underwent more and faster trafficking into tissues while effector memory and terminal effector T cells were more likely to remain in circulation. In SIV+ and aged animals, we observed increased trafficking of all subsets into tissues. A possible result of increased trafficking is that naïve and central memory cells may spend less time in lymphoid tissue and may therefore be less likely to initiate an immune response. Increased trafficking of effector cells may result in cytotoxic cells entering tissues and potentially increasing inflammation, a hallmark of the immunosenesence found in both aged and HIV+ patients. Using SIVS to evaluate trafficking in aged and SIV+ rhesus provides new insights into the crucial role of lymphocyte trafficking in the immune system.

Supported by Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Disease, National Institutes of Health