bluuebunny/crossref_metadata_2025_split huggingface dataset:
Defining the features facilitating accurate prediction of antibodies binding to the same epitope
Authors: Jarjapu Mahita , Peters Bjoern 
Published: August 2023

Abstract
Antigen specificity of antibodies is defined mainly by their complementarity-determining regions (CDRs) which show high variability in their sequences and structures. Antibodies having identical CDR sequences typically bind to the same epitope on an antigen. The conventional paradigm is that the function, including the epitope specificity, of an antibody is determined by its structure. However, we wanted to estimate how much additional information is contained in an antibody’s structure, relative to its sequence alone. High-throughput sequencing (HTS) technologies and advances in structural determination methods have led to an increased amount of antibody sequence and structural data in recent years. Here, we set out to leverage the large body of publicly-available antibody data to identify features that are most informative in predicting if two antibodies are likely to bind to the same epitope. We utilized three datasets of antibody pairs to develop machine learning (ML) models for investigating the contributions of sequence and structural features in predicting if two antibodies are likely to target the same epitope. Sequence features included pairwise sequence identities, while structural features included CDR structural similarity as well as the geometrical similarity of antibody paratopes. Surprisingly, features based on CDR sequence identity alone seemed to be sufficient to predict whether a pair of antibodies bind to the same epitope. Inclusion of CDR or paratope structural information in addition to CDR sequence information did not lead to major improvements in the predictive performance of the ML models. These findings suggest that antibody CDR sequences retain most of the information about antigen specificity.
National Institute of Allergy and Infectious Diseases of the National Institutes of Health
Immunogram: a Generalized Framework to Measure a Person’s Immune System
Authors: Medetgul-Ernar Kwat , Mohsin Azam , Davis Mark M 
Published: August 2023

Abstract
“How is my immune system?” This is a simple question, but there is no metric to assess the overall performance of the human immune system. Current clinical methods that evaluate a person’s immune system only provide limited information, like cell counts or immunoglobulin levels, and fail to capture the global immune state. This project aims to develop Immunogram, a generalized framework to measure a person’s immune system. Immunogram is a Julia-based software that links cells, proteins, and other molecules in a knowledge graph. Immunogram delineates subgraph patterns that describe human immune states and uses them to characterize a person’s immune system. Immunogram can be used to identify immune cell types, predict vaccine response, and determine if a person has an ongoing infection.
Kwat Medetgul-Ernar was supported by Stanford University Medical Scientist Training Program grant T32-GM007365 and T32-GM145402.
Modeling the SARS-CoV-2 receptor ACE2 indicates that protein-bound Zn affects distant protein stability and interactions
Authors: Fatouros Peter R , Roy Urmi , Sur Shantanu 
Published: August 2023

Abstract
Molecular Dynamics (MD) is a widely used drug design tool capable of rapidly screening the binding capacity of many candidate inhibitors in a cost-effective manner. This approach has been leveraged to search for molecules that can bind to angiotensin converting enzyme 2 (ACE2) and prevent SARS-CoV-2 viral entry to host cells. However, ACE2 is difficult to model due to an embedded zinc ion (Zn), which introduces complex charge transfer and polarization. Since the embedded Zn is distant from the site of ACE2 binding, many groups have assumed Zn does not impact protein interactions and have excluded Zn while modeling ACE2. Here, we evaluated the impact of Zn by performing MD simulations of Zn-bound and Zn-free versions of the ACE2-spike protein (S1) and ACE2-monoclonal antibody (mAb) systems . We found that excluding Zn had a significant effect on total protein stability and the stability of interacting residues, indicating that Zn can impact distant protein sites. Additionally, we discovered that including protein-bound Zn in MD simulations improved the binding free energy of both systems by −3.26 and −14.8 kcal/mol in the ACE2-S1 and ACE2-mAb systems, respectively. These data suggest that excluding Zn may alter inhibitor selection decisions and should be included for accurate modeling. Interestingly, ACE2-mAb is particularly sensitive to changes in receptor structure, likely reflecting its generation against in vivo Zn-bound ACE2. Collectively, our data demonstrates that excluding Zn significantly impacts MD simulation outcomes which inform inhibitor selection, particularly when modeling receptor-mAb complexes.
Comparison of CAR-T cell-mediated cytotoxicity assays with suspension tumor cells using high-throughput plate-based image cytometry method
Authors: Chan Leo , Sun Yu-Jun , Chen Yi-Chun , Hua Wei-Kai , Wu Sariena 
Published: August 2023

Abstract
In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Traditionally, in vitro CAR-T cell-mediated cytotoxicity is assessed using release assays such as 51Cr (radioactivity), calcein (fluorescence), and LDH (enzymatic). Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo high-throughput plate-based Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. Performing time- and E:T ratio-dependent CAR-T cell-mediated cytotoxicity assays, the GFP-based method demonstrated higher sensitivity in detecting the level of cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions.
None
A novel approach for identification of highly potent pathogen specific human CD8 T cells from peripheral blood
Authors: Anikeeva Nadia , Sykulev Yuri 
Published: August 2023

Abstract
Measuring frequency and quality of antigen-specific T cells serves as essential indicators for diagnostic and prognostic outcomes of the immune response. We have developed a novel assay allowing to measure the frequency and quality of individual antigen-specific T cells within polyclonal population of CD8 T cells assembled into tight monolayer. The assay determines kinetics and pattern of Ca2+ mobilization in every individual cell. Those T cells demonstrating rapid and sustained Ca2+ influx are highly potent and capable to rapidly destroy target cells. This has been confirmed by testing responses of HIV-specific CD8 T cells against variants of HIV-derived peptide ILKEPVHGV that induce responses of different potency. These data were consistent with comparison of HCMV-specific CD8 T cells from healthy donors and people demonstrating HCMV reactivation followed bone marrow transplantation. Vast majority of the CD8 T cell from healthy adults revealed rapid and sustained Ca2+ response. HCMV-reactivation was associated with a large percentage of CD8 T cells demonstrating oscillating Ca2+ mobilization while fraction of the T cells showing rapid Ca2+ response was very small. Our goal is to isolate potent T cells and sequence their genes encoding TCR that could be utilized for engineering of highly potent T cells to attack pathogens and cancer cells.
Supported by grants from NIH (U01 AI148117)
T cell functional phenotyping on a single cell scale using hydrogel microparticles
Authors: Kizerwetter Monika , Koo Doyeon , Di Carlo Dino , Spangler Jamie B 
Published: August 2023

Abstract
Engineered cell therapies, especially chimeric antigen receptor (CAR) T cell therapies, have shown much promise in treating various cancers, most notably hematologic malignancies. However, the success of these therapies is highly variable between cancer types and across patient populations. A growing body of literature suggests that certain T cell functional properties are key drivers of response; thus, it is imperative to develop a more thorough understanding of the functional heterogeneity in T cell populations in order to inform the design of the next generation of cancer immunotherapies. Here, we used a new technology our team developed known as nanovials, which are microfluidically manufactured hydrogel microparticles, to enable the specific activation and capture of secreted products of T cells on a single-cell level. Leveraging this platform, we characterized the secretion of IFN-γ and TNF-α by individual CD8+ T cells harvested from OT-I mice. After initial expansion after isolation, T cells were loaded into functionalized nanovials and specifically activated with OVA peptide. The cells loaded in the nanovials were then separated based on cytokine secretion levels via fluorescence-activated cell sorting (FACS). Unlike traditional cytokine secretion assays which require fixation and permeabilization, cells remain viable throughout our process, allowing for sorted subpopulations to be dissociated from the microparticles, expanded, and further analyzed via cytotoxicity, co-culture, or phenotypic studies. Collectively, our efforts introduce a new paradigm for elucidating the functional properties of immune cells, providing critical insights that will guide the future design of engineered cell therapeutics.
M.K. is a recipient of a National Science Foundation Graduate Research Fellowship Program award.
Characterization of germinal center and circulating T follicular helper cells using both intracellular and surface CITE-seq
Authors: Cashman Siobhán B , Swift Teresa , Song Hye-Won , Wright Adam , Ayer Aruna 
Published: August 2023

Abstract
T follicular helper (TFH) cells are a CD4+ T cell subset that play a critical role in the formation of germinal centers (GC). TFH cells are essential to produce protective antibody responses through regulation of clonal selection & differentiation into activated antibody-secreting & memory B cells. TFH cell differentiation is a multistep process that begins with presentation of antigen by dendritic cells to naïve CD4+ T cells, where CD4+ T cells commit to the TFH lineage by expressing the lineage defining transcription factor B cell lymphoma 6 (Bcl-6). TFH cells are phenotypically characterized by high expression of C-X-C chemokine receptor 5 (CXCR5) & programmed cell death-1 (PD-1) on the cell surface. CXCR5+ memory CD4+ T cells present in peripheral blood represent a circulating TFH (cTFH) cell subset. In contrast to TFH cells, cTFH cells may express low levels of Bcl-6 with numerous reports of subsets that differ both phenotypically & functionally. Here we characterise the heterogeneous subsets of TFH & cTFH cells using a combination of intracellular CITE-seq, surface CITE-seq and whole transcriptome single-cell RNA sequencing (scRNA-seq) analysis. Germinal center and circulating TFH cells were enriched by sorting memory CD4+, CXCR5+, PD-1+, and memory CD4+ CXCR5+ cells, respectively. We examined the expression of Bcl-6 & other transcription factors, using protein and RNA expression data, across pre-TFH, TFH & cTFH cells. Together, these data demonstrate how a combination of intracellular CITE-seq, surface CITE-seq & scRNA-seq may be used to deepen our understanding of the differentiation states & subsets of cells present at low abundance, by enabling detection of intracellular and surface protein along with high-quality mRNA.
Design and Validation of Ultrahigh-plex Discovery Panels for Immune Checkpoint Targets
Authors: Duerrschmid Clemens , Mukherjee Avik , He Yan , Berry Shannon , Shang Olive , McLane Michael , Ihley Nicholas , Zheng Yi 
Published: August 2023

Abstract
Ultrahigh-plex single-cell spatial phenotyping is revolutionizing immuno-oncology research. To enable in-depth characterization of the tumor microenvironment (TME), we have developed and validated pre-optimized antibody panels on cancer tissues to reveal key cell types and cellular architecture using the PhenoCycler™-Fusion system.
We designed multiple panels that answer specific biological questions about the TME, and once multiplexed together create a comprehensive view of the structure and function of the TME. The resulting PhenoCode™ Discovery panels include markers that identify immune cell types, activation states, immune checkpoints, and mediators of proliferation. Each antibody in the panel was conjugated to an oligo barcode and stained on human FFPE tissue. Staining specificity of each conjugated antibody was assessed by comparing immunofluorescent results from PhenoCycler™ with a DAB chromogenic immunohistochemistry assay. The slide stained with a PhenoCode™ Discovery panel was imaged on a PhenoCycler™-Fusion platform, where dye-labeled oligo reporters (complementary to the barcodes) are hybridized to the barcode to visualize the antibody. Antibody concentration, exposure time, and corresponding dye were further optimized, verified, and validated as a whole module using tonsil and cancer tissues based on image analysis and intensity analysis.
In this study, we showcased the rigorous process used for development and validation of PhenoCode™ Discovery panels and demonstrated how they can work together to collect comprehensive data from one tissue sample. The introduction of these pre-optimized antibody panels marks a pivotal point in the evolution of ultrahigh-plex spatial phenotyping.
Multi-pass flow cytometry using laser particle barcodes for easy high-marker analysis
Authors: Kwok Sheldon J , Hyun William , Forward Sarah , Fahlberg Marissa , Abbott Geoffrey , Assita Emane Rose , Zhu Tony 
Published: August 2023

Abstract
Current flow cytometry requires samples to be fully processed and stained for all markers of interest before acquisition. However, assay development become increasingly difficult when many colors (&gt;10) are used at once due to spectral spillover. Assay complexity is hampered further by markers that require special cell processing and restrict the choice of fluorophores or antibodies.
We have developed a method to dramatically simplify high-marker flow assays by breaking down a complex measurement into multiple, easier measurements. Our method involves optically barcoding individual cells with semiconductor-based laser particles (LPs) that emit laser light in the infrared. Using LP barcodes and a custom flow cytometer, we track millions of cells across repeated measurements or passes. Cells are de-stained and re-stained after each pass.
We demonstrate several applications of multi-pass flow cytometry. First, we show deep immunophenotyping of human PBMCs, measuring 33 markers with 13 colors over 3 cycles. We validate our method against state-of-the-art, reproducing expected cell types and showing improved data quality due to significantly reduced spillover. Second, we show deep characterization of stimulated mouse T cells, in which an unprecedented 32 markers including activation markers, intranuclear proteins, and intracellular cytokines are detected on the same cell type. Finally, we measure cells before and after harsh cell processing steps to recover signals that would otherwise be lost, such as concurrent detection of cytoplasmic GFP and cell cycle.
Multi-pass flow cytometry reduces the barrier to high-marker analysis by simplifying complex panels and enabling detection of sensitive signals without compromise.
Supported by NIH grant R44GM139504
StarBright Dyes:-Making Panel Design Easier For Everyone
Authors: Blundell Michael , Sanderson Sharon 
Published: August 2023

Abstract
Following simple rules during multicolor panel building can help alleviate some common problems when immunophenotyping common immunological samples. However, building large panels can still be challenging.
Bio-Rad’s new StarBright Dyes have been specifically designed for use in multicolour conventional and spectral flow cytometry to help alleviate common challenges such as brightness, spillover, fixation and buffer compatibility. Data we show here reveals how StarBright Dyes can help make panel design easier for everyone.
Longitudinal somatic mutation analysis of individual human CD8+ T cells by a UMI-based single cell mutation detection (USCMD) method
Authors: Hemani Humza , Ahmad Raheel , Cifello Jeffrey , Li Siyi , Li Jiangyuan , Lu Jian , Ellabaan Mostafa , Weng Nanping 
Published: August 2023

Abstract
Somatic mutation is a contributing factor for cancer and aging. DNA-based single-cell mutation detection methods have been developed, but are hindered by the limited number of cells examined as well as by errors from PCR amplification and sequencing methods. Here, we developed a UMI-based scRNAseq mutation detection (USCMD) method that allows for the examination of up to 10,000 cells per sample with reduced PCR and sequencing errors, and which also filters potential errors of RNA polymerase II. We applied USCMD to analyze the scRNAseq data from human CD8+ T cells collected from 8 donors with 2 visits over a period of 9 years (aged from 30 to 69 years at first visit). We found that some novel missense mutations were associated with changes (increase or decrease) in the number of cells carrying them over time, and were able to identify clonally related cells based on shared somatic mutations. Together, this USCMD method improves the accuracy of mutational analysis of scRNAseq data and allows parallel transcriptional assessment of mutated and unmutated cells to understand their potential functional consequences.
Intravital Longitudinal Three-Photon Imaging of Lymphocyte Migration in the Mouse Spleen
Authors: Choe Kibaek , Xu Chris 
Published: August 2023

Abstract
For the last 20 years, intravital two-photon (2P) imaging has been a powerful tool to visualize lymphocyte dynamics in mouse lymphoid organs such as bone marrows and lymph nodes. Although spleen is a key lymphoid organ for adaptive immunity against blood-derived antigens, the lymphocyte dynamics in the spleen are poorly understood because intravital 2P imaging of spleen is challenging. Visualizing the white pulp in the spleen where most lymphocytes are located requires the excitation light to propagate through the red pulp which contains a lot of blood, causing high light-scattering. In addition, intravital spleen imaging suffers from motion artifact due to the mouse breathing motion. To overcome these challenges, we applied three-photon (3P) microscopy for spleen imaging, which can reduce light scattering and improve image contrast in deeper regions compared to 2P microscopy. We also designed a new implantable, chronic imaging window for reducing spleen motion in our up-light microscope. We could identify white pulp regions by in vivo labeling of the marginal zone of white pulp. Using 3P microscopy at 1300 nm wavelength, we could visualize EGFP+ lymphocytes in white pulps at approximately 400–500 μm depth below the surface while 2P microscopy at 920 nm could only reach imaging depths of 160–250 μm in the same region. We then determined the temperature of the imaging window that is required for maintaining normal lymphocyte motility during imaging by monitoring the lymphocyte motility at different temperatures. This intravital longitudinal 3P imaging technique has the potential to examine lymphocyte dynamics in blood-derived antigen-induced inflammatory process in the spleen.
A novel method of digital ELISA using single molecule amplification enables sub-attomolar detection limits for blood-based proteomics and biomarker measurements
Authors: Lambert Jeremy 
Published: August 2023

Abstract
The application of measuring low-abundance protein biomarkers in blood (serum or plasma) can often present challenges due to limits of sensitivity or precision. Digital proteomics technologies such as the digital enzyme-linked immunosorbent assay (ELISA) have enabled 1000-fold increases in sensitivity over conventional immunoassay method. However, current digital ELISA technologies can lack sufficient sensitivity to quantitatively measure many low abundance proteins in blood samples.
We report the development of digital enzyme-linked immunosorbent assays (ELISAs) based on single molecule amplification with improved sensitivities over conventional digital ELISA, enabling detection of proteins at sub-attomolar concentrations. The improvements in sensitivity were based on using fewer beads to capture the target proteins (5000 vs. 500 000 beads) that increased the ratio of molecules to beads, and increasing the fraction of beads that were analyzed (bead read efficiency). Using this approach, we characterize limit of detection (LOD) of of 10–100X more sensitive than the standard digital ELISA. A digital ELISA with improved sensitivity was used to measure cytokines in serum and plasma samples with values consistently above the assay lower limits of quantitation (LLoQ). The results were consistent with a kinetic model of binding that showed that combining capture antibodies with high on-rates with high antibodies per bead yields the greatest improvement in sensitivity. The novel approach to multiplex single molecule proteomics with attomolar sensitivity provides the potential to accelerate insights in proteomics biomarkers research, clinical applications and diagnostics.