bluuebunny/crossref_metadata_2025 huggingface dataset:
CIS inhibits Treg-intrinsic TH2 program and suppresses allergic airway inflammation
Authors: Yang Xuexian O , Zheng Handong , Wu Xiang , Jiang Ruo-Lan , Castillo Eliseo , Chock Cameron , Zhou Qin , Liu Meilian , Dong Chen
Published: January 2023

Maintenance of regulatory T (Treg) cells is crucial for the regulatory function of Treg cells in immune homeostasis and self-tolerance; however, the detailed mechanisms remain elusive. In this study, we found that the cytokine suppressor CIS is required for maintenance of Treg identity. Mice with Treg specific Cis-deficiency displayed aggravated experimental allergic asthma and in adulthood, developed splenomegaly, lymphadenopathy and spontaneous eosinophilic airway inflammation, accompanied by accumulation of memory T helper (TH) cells. Cis-deficiency led to decreased expression of Foxp3 and suppressive function of Treg cells. Cis-deficient Treg cells expressed TH2 signature genes, Gata3, Irf4 and Il4, and excessive IL-4-STAT6 signals resulted in repressive chromatin modification in the Foxp3 locus and permissive modification in the Il4 loci. In vitro, blockade of IL-4 restored expression of Foxp3 and suppressive function of iTreg cells. Thus, we identified a novel feedback loop in stabilization of Treg cells and inhibition of TH2 type inflammation.
In vivo induction of T regulatory cells with recombinant mouse IL-2_anti-mouse IL-2 antibody complex
Authors: Soto Hortensia , Moyron-Quiroz Juan , Moya Zoila R , Tam Miguel , Love James M , Yang Xifeng , Nguyen Dzung , Zhou Jie
Published: January 2023

T regulatory cells (Tregs) control peripheral tolerance, and their immunosuppressive function is essential to maintain a balanced immune system. Tregs constitutively express IL-2Rα (CD25) and the transcription factor FoxP3. IL-2 is a pleiotropic cytokine and is vital for the development of Tregs. IL-2 binds to the trimeric receptor formed by IL-2Rα, IL-2Rβ (CD122), and the common gamma chain IL-2Rg (CD132). In this study we induced in vivo Treg (CD25bright, FoxP3+, CD4+) expansion by injecting a complex made of recombinant mouse IL-2 and anti-mouse CD25 (clone JES6-1A12). The mice were injected for four consecutive days with different concentrations of IL-2_JES6-1A12 complex, as well as IL-2_JES6-1A12 complex plus an excess of IL-2, or IL-2 alone. There was a significant induction of Tregs in mice that received the complex as compared to animals that received only a comparable dose of IL-2 alone. The ratio Tregs/effector cells (defined as CD8+,CD44+, CD122+) was approximately two fold in mice that received the complex. Mice injected with the complex plus an excess of IL-2 showed similar proportions of Tregs and effector cells. Previous studies showed that the use of a different IL-2 neutralizing clone (clone S4B6, recognizes and block CD122) preferentially induced effector cells. This experimental model will facilitate the study of diseases or immune mechanisms where Tregs mediate an immune response or immune system dysregulation.
Memory B cell recruitment to the vaginal mucosa is required for luminal antibody secretion
Authors: Oh Ji Eun , Iijima Norifumi , Iwasaki Akiko
Published: January 2023

Antibodies secreted into the mucosal barriers serve to protect the host from a variety of pathogens, and are the basis for successful vaccines. In type I mucosa such as the intestinal tract, dimeric IgA secreted by local plasma cells is transported through polymeric Ig receptors (pIgR). However, due to the paucity of pIgR and plasma cells, how and whether antibodies are delivered to the type II mucosa represented by the lower female reproductive tract (FRT) lumen remains unclear. Here, using genital herpes infection in mice, we show that primary infection does not establish plasma cells in the lamina propria of FRT. Instead, upon secondary challenge with herpes simplex virus 2 (HSV-2), circulating memory B cells that enter the FRT serve as the source of rapid and robust antibody secretion into the FRT lumen. CD4 tissue-resident memory T cells (TRM) secrete interferon gamma (IFN-γ), which induces expression of chemokines including CXCL9 and CXCL10. Circulating memory B cells are recruited to the vaginal mucosa in CXCR3-dependent manner, and secrete virus-specific IgG2b, IgG2c and IgA into the FRT lumen. These results reveal circulating memory B cells as a rapidly inducible source of mucosal antibodies for the FRT.
Single-cell RNA-seq of young and aged alveolar macrophages
Authors: Wu Yue , Zhu Bibo , Goplen Nick G. , Zhang Ruixuan , Li Ying , Cardani Amber N , Braciale Thomas J , Sun Jie
Published: January 2023

Alveolar Macrophages (AMs) are a unique population of innate immune cells that reside in the alveolar space and contribute to accommodate the ever-changing need of the lungs against the external environmental challenges. AMs derive from the fetal origins and are capable of maintaining themselves through self-renewal without the input of hematopoiesis during the adulthood. However, little is known about how aging is influencing the molecular characteristics and heterogeneity of the AMs during homeostasis. Using single-cell RNA sequencing (scRNA-seq) and Seurat clustering, we discovered AMs can be subdivided into different “subsets” with unique molecular characteristics during homeostasis. Interestingly, when we further investigate the AM population with the combined data from the young(~8w) and old(~2y) mice, the data suggested that the proliferating cell fall in the same cluster with reduced number in the old-mice group, while the poised inflammatory cells showed distinct molecular patterns between the group with different age. We hypothesize that the inflammatory and the self-renewal AM subpopulations may be maintained by distinct extracellular signals. With the progress of aging, the self-renewal ability of AMs would reduce because of the number of proliferating cells but not the change of molecular patterns. At the same time, the inflammatory population would change its characteristics with age, and potentially lead to functional change upon activation. Further validation of the AM subset identity as well as the developmental and functional interrelationship of these subsets are required. In addition, the extrinsic and intrinsic signals responsible for the maintenance of these AM subsets are to be explored.
Multiparametric analysis of cell health status using flow cytometry
Authors: Low Quentin , Calderon Veronica , Montoya Leticia , O'Grady Mike
Published: January 2023

Cellular responses to drug treatment can be varied and heterogeneous. Often, a multiparametric approach is necessary to identify cellular pathways that are responding or affected. To maximize the number of parameters analysed per sample, we aimed to combine several functional sensors into one assay. In addition, we set out to streamline the workflow focusing on live cell responses. To this end, we designed a flow cytometry-based detection system centered on a single incubation condition for sensors that have minimal spectral overlap. Analysing Jurkat cells treated with camptothecin, a topoisomerase inhibitor that triggers cellular apoptosis, we were able to simultaneously detect the loss of cells in G2/M phase using the stoichiometric DNA dye, Hoechst 33342; an increase in apoptotic cells with a sensor for activated caspase 3/7 (CellEvent Caspase-3/7 Green); the loss of mitochondrial membrane potential with tetramethylrhodamine, methyl ester (TMRM); and an increase in dead cells as well as cells in late apoptotic stage, using a SYTOX viability dye. We also tested another four-sensor combination that focused on detecting cellular stress. Upon prolonged camptothecin induction, we were able to detect an increase in mitochondrial stress with MitoSox, a sensor that detects mitochondrial oxidative stress; an increase in cellular stress with a CellROX reagent specific for reactive oxygen species. There was also a loss in mitochondrial membrane potential as well as a greater loss of cellular viability. In summary, using a model of cellular response to drug treatment, we performed multiparametric cell status analysis with a combination of cellular detection reagents on a flow cytometer.
A Robust and Scalable Method to Crosslink Antibodies and Proteins
Authors: Zhao Qin , Patel Deven , Wu Haiyan , Liao Jinfang , Diwu Zhenjun
Published: January 2023

The conjugations of biopolymers are critical tasks in life science field. A traditional method is to use SMCC, but SMCC-modified protein is unstable and easy to cause self-crosslink if the protein contains thiol group. Another known method is based on S-HyNic and Sulfo-S-4FB chemistry, but catalyst is required and the procedure is tedious and long.

We developed a Buccutite method to crosslink antibodies to biopolymers (e.g. enzymes and ligonucleotides) or nano particles (such as Qdots and latex beads). This new technique is based on a pair of crosslinkers: Buccutite FOL and MTA. One protein is labeled with MTA, and the other is pre-activated with FOL; when two parts are mixed, the conjugation occurs promptly without any catalyst required.

The Buccutite method was validated with the conjugation of antibody with HRP or phycobiliproteins (PE, APC or their tandems). ELISA assays showed the Buccutite antibody-HRP has the same sensitivity as traditional antibody-HRP conjugate. Flow cytometry result also showed the Buccutite conjugates performed as well as the ones prepared with traditional SMCC method when evaluated with human lymphocytes CD antibodies conjugates. Pre-activated proteins (e.g., PE-FOL) were stable for 12 months without any decreasing in performance. Buccutite conjugation was very efficient: there was less than ~10% of the free antibody left, while there was still more than 40% free antibody remaining for the traditional SMCC method.

In conclusion, Buccutite method has demonstrated a way to prepare conjugate at high efficiency in short reaction time and mild reaction conditions with the same performance. It is a superior and effective alternative to the existing methods for crosslinking biopolymers to their targets.
Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function
Authors: Shifrut Eric , Carnevale Julia , Ashworth Alan , Marson Alexander
Published: January 2023

Human T cells are central effectors of immunity and cancer immunotherapy. CRISPR-based functional studies in T cells could prioritize novel targets for drug development and improve the design of genetically reprogrammed cell-based therapies. However, large-scale CRISPR screens have been challenging in primary human cells. Critical biology of human immune cells, including key signaling pathways and effector functions, may not be recapitulated in immortalized cell lines. We developed a new method, sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes characterized hits and identified perturbations that enhanced cancer cell killing. SLICE coupled with single-cell RNA-Seq revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling responses to adenosine signaling. In summary, we have developed a novel pooled CRISPR screening technology with the potential to explore unmapped genetic circuits in primary human cells and to guide the design of engineered cell therapies.
Novel plate-based detection method for T cell activation/proliferation, migration, and cytotoxicity assay using image cytometry
Authors: Chan Leo
Published: January 2023

The field of cancer immunotherapy has grew significantly due to numerous successful trials in cancer vaccines, antibody-based treatments, CAR T cell therapy, and other immunotherapies. Specifically for T cell-based assays, they have been traditionally analyzed using ELISA or ELISpot to detect specific cytokines concentration for measuring T cell responses. Other methods such as T cell activation/proliferation, migration, and cytotoxicity assays have employed flow cytometry or microscopy to understand the functionality of T cells. In the recent years, image cytometry has been used to develop robust assays to monitor T cell immune response, which is highly critical for immuno-oncology research.

In this work, we demonstrate the use of Celigo Image Cytometer to perform high-throughput in vitro T cell-based functional assays. Three direct cell counting methods are developed for label-free T cell activation and proliferation measurement, T cell migration assay, as well as T cell-mediated cytotoxicity. First, primary T cells are seeded into a 96-well plate and stimulated with antibodies or co-culture with cancer cells. The cells are directly counted in the well over 48 hours in bright field images. Next, T cells are seeded in a transwell with chemokines in the bottom wells. As the T cells migrate through and drop to the bottom, the cells are directly counted using bright field or fluorescence with Hoechst staining. Finally, T cell-mediated cytotoxicity assay is performed by co-culturing CD8 T cells with calcein- or fluorescent protein-labeled cancer cells. As target cancer cells are killed, the fluorescence dissipates, and by counting the changes in the number of “live” target cells, the cytotoxicity percentages are measured.
The “Crisis of Reproducibility” of Animal Studies: Causes and Solutions
Authors: Shollenberger Lisa M , Hentze Hannes , Ibsen Eric
Published: January 2023

A majority of post-phase III clinical trial therapies have historically failed to receive regulatory approval due to a lack of demonstrated efficacy. According to a 2012 study published by Amgen researchers, 90% of the 50+ in vivo oncology studies were irreproducible. Over the last two years, questions and debates concerning the validity of the preclinical science data have resulted in multiple published reviews.

Suboptimal disease models, inadequate or inappropriate controls, and poor documentation practices (including electronic spreadsheets) will be discussed as primary causes of irreproducibility in animal research. To address these concerns, several best-practice resources and initiatives have been made available to the research community. The ARRIVE guidelines, PHISPS protocols, and the N3R Design Assistant will be described as practically-applicable concepts and publicly-available resources to address issues contributing to the “Crisis of Reproducibility”.

Researchers have increasingly adopted animal study workflow software applications and databases, which were developed to address this crisis. By applying such automated and time-saving technologies, researchers can now more easily standardize the highly variable but detailed processes of study design, procedure adherence and descriptions, animal welfare parameter monitoring, graphing, data analyses, report creation, task and measurement scheduling, and data preservation. Using some real-life examples, the advantages and disadvantages of preclinical study software applications and the use of electronic spreadsheets will be discussed in the context of helping to resolve the “Crisis of Reproducibility” in animal studies.
Adarza’s Ziva technology platform enables highly multiplexed label-free detection of serum analytes
Authors: Schmuke Jon J , Mueller Ernie , Darby Amanda , Wente-Roth Traci , Vohra Naila , Haley Jeaneen , Hodge Tracey , Pillers Michelle
Published: January 2023

New multiplex, label-free methods for the sensitive and quantitative detection of serum proteins are highly desirable, as they have the potential to dramatically accelerate biomedical research in the age of multi-omic, big data driven personalized medicine. We have used Arrayed Imaging Reflectometry (AIR™), a label-free protein microarray technology that relies on the target binding-induced perturbation of an antireflective coating on the surface of a silicon chip, to create a multiplex antibody array for the detection of human serum cytokines and inflammatory markers. The chip is able to provide quantitative information about protein concentrations, with limits of detection in picograms per mL. Provided in a standard 96-well microplate format, the label-free nature of the array will permit rapid expansion in plex size and enable simple, flexible creation of custom panels as there are no secondary antibodies or labels to cross-react. Here we demonstrate validation parameters of sensitivity, precision, and accuracy for inflammatory markers (>20) in a new multiplex technology suitable for analyzing trends across cytokine and inflammatory marker profiles comparable to traditional ELISAs. With array capabilities from low to hundreds of plex combined with a hands-free, automation-friendly workflow enabled by the Ziva instrument, the technology has been designed for the ultimate user-experience.
Optimum processing conditions for single cell RNA sequencing on frozen human PBMCs
Authors: Hanamsagar Richa , Marcus Robert , Chamberlain Mathew , de Rinaldis Emanuele , Savova Virginia
Published: January 2023

The field of single cell RNA sequencing (sc-SEQ) has exploded in the past few years. From picking up single cells manually under a microscope, to droplet-based encapsulation of cells using microfluidics – this technology has improved in leaps and bounds. Common droplet-based technologies include inDrop, Drop-seq and 10X Genomics Chromium. All three technologies utilize microfluidics for encapsulating single cells & uniquely barcoded beads within an oil droplet. They differ in their bead material/manufacturing, barcode design and the range to which their operation can be customized by the end user. However, the performance of each sc-SEQ each technology is dependent on factors such as ability to obtain pure, viable single-cell suspension, and ability to accurately quantify the number of cells before running them through the machine. Here, we compare and contrast different conditions for cell processing that can affect single-cell sequencing results – including cell counting and purifying methods, as well as cell subtype enrichment kits; followed by single cell encapsulation, library preparation and analysis using 10X Genomics Chromium workflow.
The contribution of tumor type, stage and antigen in the Antibody-Antigen kinetics by ELISA in adult and pediatric cancer patients
Authors: Marquez-Manriquez Juan P , MATUTE-BRISENO JOSE ANTONIO , CAMACHO-HERNANDEZ ALEJANDRO , ICEDO-ZAMORA SALVADOR , Alejandro Lucero-Diaz PEDRO ALEJANDRO
Published: January 2023

Background

Most of the ELISA studies in cancer patients use different source of antigens, which is mainly limited to recombinant proteins produced in E. coli and this may impact the global results between the studies using even the same antigen because most of the researchers assume that all the tumor types and all the antigens would behave equally, which we believe is not well established immunologically and clinically.

Methods

The repository bank was used (n=75 adults) and (n=35 pediatric) and (n=90) presumably healthy patients. The serum was analyzed by ELISA at different times starting at 1, 2, 4,6,12,24, 48 and 72 hours where the serum was placed at 1:200 dilution with a panel of 8 recombinant, purified proteins and predicted immunogenic peptides from all the 8 proteins. We correlate the kinetics of antibody-antigen binding with several clinical parameters such as stage, PFS, ECOG and the level of some acute phase proteins.

Results

We demonstrated that the optical density (OD) at 650 nm of the cancer patients is different depending of the tumor stage (p=0.0001) and age as in in pediatric tumors we found statistical significance in the antibody-binding with respect to the adult tumors (p=0.0005) in 2 hours versus 72 hours. Importantly, we found that the patients with early binding at 2 hours had better prognosis (mOS=19 months) than those at 72 hours (mOS=7 months). Also the best antigen to demonstrate the antigen specific immune response in cancer patients was the purified proteins (p=0.0001) and predicted peptides (p=0.001).

Discussion

This data demonstrate that the nature of the antigen, the kinetic of exposure of the antibody-antigen impacts the results and the clinic correlation with several parameters including tumor stage.
Optimizing the pharmacokinetic properties of polymeric IgA in mice
Authors: Rajan Sharmila , Lombaba Noelle Twyla , Mandikian Danielle , Boswell Andy , Yip Victor , Bravo-Perez Daniel , Chen Eugene , Wang Jianyong , Phung Wilson , Lee Sophia , Farahi Farzam , Estevez Alberto , Gill Avinash , Sandoval Wendy , Viajar Sharon , Ciferri Claudio , Matsumoto Masrissa , Spiess Christoph
Published: January 2023

IgA antibodies have broad potential as a novel therapeutic platform based on their superior receptor-mediated cytotoxic activity, potent neutralization of pathogens, and ability to transcytose across mucosal barriers via polymeric immunoglobulin receptor (pIgR)-mediated transport, compared to traditional IgG-based drugs. However, the transition of IgA into clinical development has been challenged by complex expression and characterization, as well as rapid serum clearance. Here, we present the biochemical, biophysical, and structural characterization of recombinantly produced monomeric, dimeric and polymeric human IgA. We further explore two strategies to overcome the rapid serum clearance of polymeric IgA, which may enable the IgA platform the potential to specifically target pIgR expressing tissues, while maintaining low systemic exposure. This strategy may thus allow for targeting antigens previously untenable with conventional IgG1, while allowing for lower drug exposure in non-targeted regions.
T cell receptor repertoire detection from dry blood spots
Authors: Li Song , Pan Wenjing , Wu Shang-Gin , Liu Hongna , Byrne-Steele Miranda , Brown Brittany E , Depinet Mollye , Hou Xiaohong , Han Jian
Published: January 2023

Immune repertoire amplification of T cell receptors (TCRs) coupled with high throughput sequencing provides detailed insight into the immune system. A common starting sample type is total RNA extracted from whole blood by venipuncture. However, the costs, participant burden, regulatory constraints, and logistics associated with venipuncture and RNA handling are major barriers to clinical application or community-based research on various diseases. This study aims to identify TCR repertoires from dry blood spots (DBS), a method that could help collect real-world data for biomarker applications. Finger-prick blood was collected onto a Whatman filter card, and RNA was extracted from DBS. Fully automated multiplex PCR was performed to generate a TCRβ chain library for next generation sequencing (NGS) analysis of unique CDR3s (uCDR3). According to statistical analysis and laboratory confirmation, forty 2-mm punch disks from the filter cards were enough to detect the shared top clones and have a strong correlation in uCDR3 discovery with whole blood. uCDR3 discovery was not affected by storage temperatures (room temperature or −20°C) or storage durations (1, 14, and 28 days) when compared to whole blood. About 74 – 90% of top 50 uCDR3 clones of whole blood could also be detected from DBS. The DBS-based TCR repertoire profiling method is minimally invasive, provides convenient sampling, and incorporates fully automated library preparation. The system is sensitive to low RNA input, and the results are highly correlated with whole blood uCDR3 discovery allowing study scale-up to better understand the relationship and mutual influences between the immune repertoire and various disease states.
Complete all-in-one immune repertoire profiling of newborn babies via novel dimer-avoided multiplex PCR (dam-PCR)
Authors: Hou Xiaohong , Pan Wenjing , Depinet Mollye , Brown Brittany E , Eisenhower Mary , Byrne-Steele Miranda , Weber Michele , Dworsky Meyer , Callison Sharon , Han Jian
Published: January 2023

There is an increased demand for immune repertoire profiling in both basic research as well as in various clinical settings, such as during vaccine development, lymphocyte tracking in minimal residual disease (MRD), hematopoietic stem cell transplant recovery monitoring, immunotherapy development, and immunological monitoring for prognosis purposes during treatment. Although next generation sequencing of the immune repertoire allows detailed, sequence-specific insight into the adaptive immune response, it usually focuses on only one or two receptor chains. One of the key challenges during immune receptor amplification is the formation of dimers, which can compete with the immune amplicons of interest during library preparation. We therefore developed a novel PCR technique, dimer-avoided multiplex PCR, that allows amplification of both BCRs and TCRs in a single, quantitative multiplex reaction. Our method generates next generation sequencing libraries for all TCR and BCR loci including TCR-beta, TCR-alpha, TCR-delta, TCR-gamma, BCR-IgH, and BCR-IgK and IgL. Here, we report the application of this method to investigate the immune repertoire of cord blood from newborn babies. Our study revealed the vast diversity and abundance of babies’ immune repertoire, the composition of TCR and BCR loci, and the sharing status of unique CDR3s among newborns. This single reaction, cost effective, all-inclusive, and quantitative immune-profiling analysis method shows promise for future applications in both basic research and clinical settings.
A pathogen-free flow cytometry based opsonophagocytosis assay protocol to quantify antibody-mediated phagocytosis
Authors: Badten Alexander Jesse , Felgner Jiin , Pone Egest J , Albin Tyler J , Fellon Corey , Kang Karen , Felgner Philip L , Burkhardt Amanda M
Published: January 2023

An important function of antibodies is their ability to mark cells for phagocytosis and subsequent degradation in a process known as opsonization. When developing new vaccine candidates, it is critical to determine whether the antigen specific antibodies elicited in response to immunization have this function. Due to the high containment working constraints of several important human pathogens, it is not always possible to directly study this property using the pathogen of interest. Therefore, we sought to develop a pathogen-free, flow cytometry based assay to quantify the opsonizing activity of antibodies elicited in response to immunization. We studied this using vaccine candidates against Coxiella burnetii, the causative agent of Q-fever that is a potential bioterrorism agent. In our assay, fluorescent polystyrene beads were conjugated to C. burnetii proteins then incubated with the antibody-containing sera. The antibody-coated beads were incubated with macrophages and phagocytosis of the fluorescent beads was assayed by flow cytometry. This assay allows for rapid quantification of the opsonizing capacity of antigen specific antibodies elicited in response to immunization.
Cis-proteomics reveals novel transcriptional regulators of Treg development and function
Authors: Feng Yongqiang , Zong Xinying
Published: January 2023

The immune suppressive function of regulatory T (Treg) cells is governed by the Foxp3-depenent transcriptional programs that remain unclear, which constraints effective manipulation of Treg behaviors for the treatment of many autoimmune diseases and cancer. To overcome the technical barriers for addressing this issue, we developed a new method to reveal the protein components of the cis-regulatory elements (cis-proteome) in Treg cells defined by Foxp3 binding, because the interaction among these DNA elements and their associated proteins ultimately determine Treg development and function. Specifically, we projected the spatial information (PSI) of Foxp3 onto its adjacent chromatins and nuclear factors with a proximity ligation reaction and identified the labeled DNA-protein complexes with chromatin immunoprecipitation (ChIP) and quantitative mass spectrometry-based proteomics.

Our Foxp3 PSI reproducibly captured majority of the open chromatins and active enhancers and promoters, representing the Treg cis-regulatory hubs, and identified 1500 proteins including many known Foxp3 interactors, crucial regulators of Treg function, and numerous novel nuclear proteins that encompass all the key steps of gene regulation. Among them 124 are highly variable in their expression levels in a cell type- or activity-dependent manner. Using a CRISPR/Cas9 approach we identified more than 50 novel nuclear factors specifically required for the induction or maintenance of Foxp3 expression.

Our study thereby offers a broadly applicable strategy to investigate the proteins involved in Treg differentiation and function and will produce many mechanistic insights into the transcriptional basis of Treg-mediated immune tolerance.
Single cell whole transcriptome analysis of disease cells to generate a targeted RNA-sequencing gene panel for the simultaneous analysis of targeted mRNA and protein
Authors: Bansal Nidhanjali , Song Hye-Won , Sa Silin , Lomas Woodrow E , Baracho Gisele V , Taylor Ian , Widmann Stephanie , Mortimer Stefanie
Published: January 2023

Single cell RNA-sequencing (scRNA-seq) is a powerful tool for understanding the sample heterogeneity of individual cells. Many methods rely on whole transcriptome analysis (WTA) to get a snapshot of the entire cellular landscape. Although WTA analysis can be used to discover novel biomarkers, this technique can be expensive. To enable scaling of experiments, WTA data can be mined to design targeted gene panels. To showcase this, we used single cell sequencing to examine thousands of B cells isolated from the bone marrow and peripheral blood of chronic lymphocytic leukemia (CLL) and healthy donors. B cells that had been sorted using the BD FACSMelody™ cell sorter were multiplexed using the BD™ Human Single-Cell Multiplexing Kit and pooled before being processed on the BD Rhapsody™ system, thereby minimizing batch effects. The resulting data was mined to design a panel of differentially expressed genes between CLL and healthy B cells that could be used for subsequent CLL phenotyping. By combining this panel with the BD Rhapsody™ Immune Response Panel (together comprising ~500 mRNAs), along with 36 DNA-barcoded BD™ AbSeq antibodies, we were able to simultaneously analyze mRNA and protein targets from a new subset of CLL and healthy B cells for additional high-resolution analysis. This study showcases the power of using WTA data to design specific gene panels that can be used alone or in combination with existing targeted panels for routine and cost-effective transcriptional profiling at a single cell level.

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

BD, the BD Logo, FACSMelody, and Rhapsody are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.
Evaluation of Cytometer Sensitivity and Stability using Automated Analysis
Authors: Bradford Jolene A , Meskas Justin , Sardina Patricia
Published: January 2023

Flow cytometers are standard tools of the immunologist, providing simultaneous measurement of many targets. The performance of a flow cytometer may vary between instruments, or over time within the same instrument. Evaluation of cytometer performance is essential to ensure results are consistent over time within the same instrument for longitudinal studies and day-to-day data consistency, and across multiple instruments when more than one cytometer is being utilized. Basic capabilities of each fluorescent detector can be estimated using the Q & B calculations, where Q is detection efficiency and B is background. Precision of measurements and minimum detectable dye can be derived from Q & B measurements, to evaluate sensitivity and stability.

In this study hard-dyed multi-level, multi-dye beads were collected in three different identically configured cytometers over 11 days. Each day data was collected in 14 different fluorescent detectors at six flow rates. An automated approach to data analysis was utilized to provide unbiased evaluation. All data files were first evaluated with flowCut to check for spurious events. Using automated gating, beads were then identified using Forward Scatter vs. Side Scatter, and flowQB was used to calculate Q & B. This data was used to evaluate intra-instrument and inter-instrument sensitivity and stability.

Results show the minimum amount of detectable dye and precision of its measurement are robust across time and different cytometers, though decreased at the highest flow rates of ≥ 500 μL/min. Using an automated unbiased approach for determining Q & B demonstrates cytometer sensitivity and consistency over time, between instruments, and across flow rates.
Developing a novel multiplexed high throughput flow cytometry based immune assay to screen kinase modulators of primary T cell activation
Authors: O’Rourke John , Gomez-Donart Andrea , Weldon Caroline , Liu Zhaoping
Published: January 2023

Modulating TCR engagement and signaling using biologics, small molecules or genetic engineering is highly relevant to many therapeutic areas. TCR signal initiation is mediated by cytosolic tyrosine kinases, leading to signal amplification through a network of serine-threonine kinases. Genetic defects, mutations and other mechanisms leading to increased kinase activity and enhanced T cell activation (TCA) is involved in many autoimmune pathologies, making kinases attractive targets for the direct inhibition of TCA and treatment of autoimmune disease.

The development of drugs and therapies regulating TCR activity require assays to profile T cell function and cell health. To address this need, we developed a novel multiplexed TCA assay based on high throughput flow cytometry technology with advanced computational algorithms for data analysis. The assay simultaneously measures T cell phenotype, time-dependent expression of T cell activation markers, cell proliferation and viability and quantitates secreted cytokines from a single 10 ml sample using a 96 or 384-well plate format.

To illustrate the value of the TCA assay in drug discovery, we performed a phenotypic screen using a 152 kinase inhibitor (KI) small molecule library for their ability to inhibit human primary TCA. Using the Profile Mapping algorithm in our ForeCyt software, we integrated 11 TCA metrics to identify multiple inhibition pathways. The Profile Map tool was used to group KI based on their inhibition phenotypes, irrespective of their known targets. These results illustrate the use of Intellicyt’s technology to deliver solutions for drug and biologics discovery.
Type II NKT cells directed immune regulatory mechanism(s) control spontaneous lupus nephritis in mice
Authors: Maricic Igor , Halder Ramesh , Marrero Idania , Kono Dwight H , Kumar Vipin
Published: January 2023

NKT cells recognize lipid antigens presented by CD1d molecules and can be categorized as iNKT cells and type II NKT cells. Type II NKT cells recognize sulfatide or lysophosphatidylcholine (LPC), and their activation results in cross-regulation of iNKT cells, tolerization of DC and control of inflammatory diseases. iNKT cells have been shown to play a pathogenic role in (NZB x NZW)F1 or BWF1 mice, a prototype for human SLE. Interestingly, anti-glycosphingolipid antibody responses have been found in lupus patients. Since sulfatide is enriched in kidney glomeruli, we have examined the role of type II NKT cells in the spontaneous development of lupus in BWF1 mice. We found that both iNKT (αGalCer/CD1d-tetramer+) and type II (sulfatide/CD1d-tetramer+) NKT cells accumulate in kidney tissues with progression of disease in BWF1 mice. Notably, iNKT cells are also activated in SLE patients and secrete the pro-inflammatory cytokine IFNg. Sulfatide-mediated activation of type II NKT cells leads to a significant inhibition of nephritis in BWF1 mice. Importantly, a clinically relevant structural analog of LPC, Miltefosine, also binds to CD1d, activates type II NKT cells and upon oral administration induces inhibition of proteinuria, anti-DNA Abs as well as infiltration of CD4+/CD8+T cells and B cells into kidney of BWF1 mice. Interestingly, the phenotype and frequency of type I interferon-secreting pDC is also altered in kidney of mice treated with Miltefosine. These studies suggest a key role for a type II NKT cell-based immune regulatory mechanism in the control of lupus and, since the CD1d-dependent pathway is highly conserved from mice to human, they have important implications for repurposing a drug for immunotherapeutic for lupus.
A nanoparticle-based approach targeting ion channels for the treatment of Lupus nephritis
Authors: Chimote Ameet A , Khodoun Marat , Duncan Heather J , Kant Shashi K , Conforti Laura
Published: January 2023

Lupus nephritis (LN) occurs in up to 60% of patients with systemic lupus erythematosus (SLE) and it remains the predominant cause of morbidity and mortality. Current available therapies for SLE have serious side effects and limited efficacy. Our research aims to develop a novel therapy for SLE with improved efficacy and minimal side effects. SLE is in large part driven by hyperactive T cells, which present with multiple defects including increased INFγ, chemokine receptors and CD40L that exacerbate tissue infiltration, inflammation and B cell stimulation. Kv1.3 channels regulate the Ca2+ signaling necessary for these effector functions. We have developed lipid nanoparticles that selectively target memory T (TM) cells and reduce their hyper-functionality by downregulating Kv1.3 via siRNAs (Kv1.3-NPs). Our in-vitro data showed that Kv1.3-NPs suppressed Ca2+ signaling, CD40L expression and IFNγ release selectively in TM cells. To demonstrate therapeutic efficacy of the Kv1.3-NPs we have developed a humanized mouse model of LN by engrafting peripheral blood mononuclear cells (PBMCs) from LN patients into immune-deficient NSG mice. Kidneys of these mice were highly infiltrated with human CD3+ T cells and these cells produced elevated IFNγ. Furthermore, these mice demonstrated a lower survival compared to control mice engrafted with PBMCs from healthy donors. Lastly, we tested the efficacy of the Kv1.3-NPs. We found that LN mice engrafted with PBMCs pretreated in-vitro with Kv1.3-NPs survived longer than mice engrafted with PBMCs pretreated with scramble RNA-NPs. We also found that Kv1.3-NPs were selectively delivered in-vivo into TM cells of humanized mice. Overall, these data support the potential therapeutic value of Kv1.3-NPs.
Investigational Treatment of Rheumatoid Arthritis with a Vibrotactile Device Applied to the External Ear
Authors: Imperato Gavin H. , Addorisio Meghan E. , Pavlov Valentin A. , Yang Huan , Diamond Betty , Tracey Kevin J. , Chavan Sangeeta S.
Published: January 2023

Rheumatoid arthritis (RA) is a debilitating polyarthritis characterized by joint tissue inflammation. Here, we assessed the effect of applying a vibrotactile device to the external ear on inflammatory responses in healthy subjects, as well as its effect on disease activity in patients with RA. We enrolled 19 healthy subjects in a randomized cross-over study, delivered vibrotactile treatment at either the external ear or gastrocnemius, and performed ex vivo whole blood assays. Vibrotactile treatment at the external ear significantly reduced TNF, IL-1β, and IL-6 levels compared to pre-treatment baseline (TNF p<0.05, IL-1β p<0.005, IL-6 p<0.005), whereas treatment at the gastrocnemius did not attenuate inflammatory responses. Vibrotactile treatment at the external ear inhibited TNF by 80% (mean ± SD: pre-treatment = 4541 ± 2721 pg/ml vs. post-treatment = 3624 ± 2810 pg/ml), IL-6 by 73% (mean ± SD: pre-treatment = 5979 ± 2094 pg/ml vs post-treatment = 4342 ± 2600 pg/ml), and IL-1β by 50% (mean ± SD: pre-treatment = 1527 ± 1429 pg/ml vs post-treatment = 765 ± 968 pg/ml) as compared to pre-treatment baseline levels. 9 patients with RA were enrolled in a prospective interventional study. Vibrotactile treatment at the external ear significantly decreased DAS28-CRP scores two days post-treatment (DAS28-CRP score mean ± SD: pre-treatment = 3.4 ± 1.4 [1.27–5.67] vs. post-treatment = 2.7 ± 0.9 [1.24–4.37], p<0.005). DAS28-CRP scores remained significantly reduced 7 days post-treatment (DAS28-CRP score mean ± SD: 7 days post-treatment = 2.4 ± 0.7 [1.21–3.68]; p<0.005). These data demonstrate that application of a vibrotactile device to the external ear attenuates systemic inflammatory responses in both healthy subjects and patients with RA.
Restraining neuroinflammation during antigen delivery as an immunosuppressive approach to prevent and treat contact dermatitis
Authors: Bandyopadhyay Mohna , Morelli Adrian E , Erdos Geza , Sumpter Tina L , Tkacheva Olga A , Shufesky William J , Falo Louis D , Larregina Adriana T
Published: January 2023

Neuroinflammation promotes the initiation and sustains chronic inflammatory disorders. Substance-P, released by sensory neurons, is the prototype neuropeptide that signals via the neurokinin 1 receptor (NK1R) to enhance cellular immunity. We observed that NK1RKO or SPKO mice do not develop contact dermatitis (CD). Hence, we hypothesized that limiting neuroinflammation during Ag entrance induces an immune-suppressive environment to prevent T cell priming and eliminate memory T cells that cause CD. We co-delivered OVA or 2,4-dinitrocholorobencene (DNCB) and two different NK1R antagonists during sensitization of a DTH reaction induced to C57/BL6 mice reconstituted or not with OTI and OTII cells. For efficient skin delivery, we generated microneedle arrays loaded with OVA or DNCB and NK1R antagonists. We demonstrate that our approach prevents the innate and adaptive immunity accounting for the initiation of CD and mitigates pre-existing pathogenic memory T cells that cause local or systemic CD relapses. Mechanistic studies demonstrate that NK1R antagonists suppress the release of pro-inflammatory cytokines in the skin, promote the death of activated CD4 Th1 and CD8 T-cells, and generate Tregs in the skin draining lymph nodes. Our data demonstrates that controlling neuroinflammation during Ag entrance prevents the generation of pathogenic effector and memory T cells accounting for the initiation and relapses of chronic inflammatory skin disorders like CD. NIH R01 AR068249 and AR071277 to ATL and LDF.
De novo DNA methylation programs regulate T cell exhaustion and limit T cell-based immunotherapies
Authors: Youngblood Ben A , Ghoneim Hazem E. , Abdelsamed Hossam Aly , Moustaki Ardiana , Fan Yiping , Crawford Jeremy , Thomas Paul Glyndwr , Stewart Elizabeth , Federico Sara
Published: January 2023

T cell-based immunotherapies have emerged as one of the most promising frontiers in the fight against cancer and chronic infections. However, it has become clear that prolonged exposure of T cells to their cognate antigen drives them toward a terminally differentiated state, limiting their capacity to mount an effector response. The commitment of T cells to this “exhausted” fate is currently a major barrier in the advancement of T cell immunotherapy efforts. To better understand mechanisms that reinforce the T cell exhaustion gene expression program, we investigated the role of de novo epigenetic programming in establishing exhausted T cells that are non-responsive during immune checkpoint blockade (ICB) therapy. Using mouse models of tumor and chronic viral infection, we observed that genetic deletion of the de novo DNA methyltransferase, Dnmt3a, in antigen-specific T cells that are chronically stimulated allowed them to remain highly functional despite expressing high levels of the checkpoint inhibitory receptor PD-1. Furthermore, PD-1 blockade treatment resulted in massive expansion of PD-1+ Dnmt3a-deficient antigen-specific T cells. Building upon the findings from our murine studies, we have generated whole-genome DNA methylation profiles of human CD8 T cells from a wide array of differentiation states, and established an epigenetic-based human T cell multipotency index. Using this index, we report that tumor-associated PD-1hi CD8 T cells from pediatric solid tumors acquire an epigenetically reinforced terminal differentiation program. Collectively, these data establish Dnmt3a-mediated de novo DNA methylation programming as a key factor in limiting CD8 T cell-based immunotherapuetic approaches.
Ex Vivo Phenotyping and Potency Monitoring of CD19 CAR T Cells With a combined Flow Cytometry and Impedance-based Real-Time Cell Analysis Workflow
Authors: Lei Ming , Ye Peifang , Lu Yan , Ji Xiaoping , Wang Andy , Li Nancy , Wang Xiaobo
Published: January 2023

Cancer immunotherapy is increasingly utilized for cancer treatment. CARs (Chimeric antigen receptors) targeting CD19 have produced strong antitumor responses in hematologic malignancies, but tumor regression has seldom occurred when CARs targeting other antigens. The state of T-cell differentiation and T cell exhaustion influences the engraftment and persistence of CAR-expressing T cells following adoptive transfer.

Here we report the use of a combined impendence-based Real-Time Cell Analysis (RTCA) and flow cytometry cell analysis workflow for ex vivo cytolytic potency monitoring of CD19 CAR T cell, as well as the examination of phenotypic and functional responses to antigen exposure over time. CD19 CAR T cell killing of HEK293 CD19 with different E:T ratio was monitored by RTCA system. Cytokine productions were measured by NovoCyte® Quanteon™ flow cytometer after 24 hrs. Co-incubation with HEK293 CD19, phenotypic and functional responses of CD19 CAR T cells towards exposure of different antigen levels were systematically examined over time. Both assays reveal that the CD19 CAR T cells show high dose-dependent killing activities against HEK293 CD19 but not against HEK293. Using the multi-color Flow Cytometry immunophenotyping, CD4 dim and CD8 dim populations are identified as effective killing cells which are enriched in central memory (CD45RA+CCR7−) cells while normal CD4+ or CD8+ T cells consist of predominant naïve-like (CD45RA+CCR7+) T cells after co-incubation with HEK293 CD19 cells. Exhaustion marker (PD-1, LAG-3, TIM-3) are overexpressed in normal CD4+ and CD8+ populations but not in CD4 dim and CD8 dim populations with CD19 antigen exposure. After antigen exposure, T cell activation levels change significantly.
CAR+ T cell anti-tumor efficacy revealed by multi-dimensional single-cell profiling
Authors: An Xingyue , Romain Gabrielle , Martinez-Paniagua Melisa , Bandey Irfan N , Adolacion Jay R T , Fathi Mohsen , Liadi Ivan , Sadeghi Fatemeh , Mahendra Ankit , Amritkar Amit , Roysam Badrinath , Singh Harjeet , Cooper Laurence J , Varadarajan Navin
Published: January 2023

T cells engineered to express chimeric antigen receptor (CAR) targeting CD19 have shown promising clinical responses in patients with certain hematologic malignancies, however, it is desirable to be able to enrich cells with enhanced anti-tumor efficacy prior to infusion. We utilized a suite of high-throughput technologies with single-cell resolution, including Timelapse Imaging Microscopy In Nanowell Grids (TIMING) that integrates cytokine profiling to reveal that persistent motility of CD19- specific CAR T cells is correlated to desirable polyfunctionality (elimination of tumor cells and cytokine secretion), contributing to anti-tumor effects. We implemented a marker-free Boyden chamber-based method to enrich CAR+ T cells with persistent motility (motile cell). Integration of transcriptomic profiling, immune phenotyping and metabolism demonstrated that motile cells are more naïve-like with higher oxidative metabolism and spare respiratory capacity. Our result also revealed that the master metabolic regulator AMP kinase (AMPK) is required for CAR+ T cells with high motility. We used a xenograft leukemia mouse model (CD19+ NALM-6) and validated that the motile cells have enhanced persistence and superior anti-cancer effect in vivo compared to the parental un-sorted population. Collectively, our multi-dimensional results demonstrated that persistent motility is a selectable biomarker of expanded CAR+ T cell bioactivity.