bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of modified peptides for live tracking of peptide-MHCII complexes during antigenspecific T cell-dendritic cell interactions
Authors: Al Souz Jafar A , Akkaya Munir , Kamdar Rahul , Kamenyeva Olena , Kabat Juraj , Shevach Ethan M , Akkaya Billur 
Published: January 2023

Abstract
The recognition of peptide-MHC class II complexes (pMHCII) presented by dendritic cells (DCs) is critical for the activation of CD4+ T cells, and proper detection of such complexes is essential for understanding T cell-DC interactions. However, current detection methods for pMHCII complexes using antibodies may block cellular interactions, limiting their use, while conventional tags, such as GFP, are too large to be loaded onto MHCII without disrupting its function. To probe pMHCII complexes, we constructed a variant of the ovalbumin peptide (OVA323–339) linked to a tetracysteine tag (CCPGCC), which can be treated with organoarsenic compounds to induce fluorescence. This modified peptide retained its functionality as it was able to stimulate T cells to the same degree as the unmodified peptide. We also analyzed signal specificity both at the pMHCII level, by using MHCII−/− APCs, and at a broader level by quenching endogenous tetracysteine expression, and confirmed that our fluorescent signal was derived from the peptide modification. We then used microscopy to visualize pMHCII movement after interaction of pulsed DCs with OVA-specific T cells in vitro and subsequently in vivo, and detected a gradual increase of pMHCII complexes at the synapse. Interestingly, we also observed a transfer of fluorescent signal from DCs to antigen-specific T cells, but not to polyclonal T cells, indicating acquisition of pMHCII, which we were able to quantify. Taken together, our results show a non-disruptive approach to assay pMHCII, and provide a window to observe intricate T cell-DC interactions in real-time.
REAlease® technology: Controlled release of antibody-fluorochrome conjugates for maximal flexibility in flow sorting applications
Authors: Evaristo Cesar , Pankratz Jennifer , Schmachtenberg Sabine , Jansen Nicole , Krauthäuser Susanne , Kinkhabwala Ali , Schlegel Karolina , Siewert Christiane , Bartholomäus Alina , Meiler Svenja , Yushchenko Dmytro A. , Dose Christian 
Published: January 2023

Abstract
Multi-parameter immunofluorescent labeling is the method of choice for the flow-based sorting of targeted cell populations from heterogeneous samples. However, existing antibody-fluorochrome conjugates used for flow sorting limit the types of downstream analyses that can be applied to the isolated cells, as their continued presence on the cells blocks specific cellular epitopes and restricts the choice of downstream fluorescence detection channels. Herein, we present a new type of antibody-fluorochrome conjugates that facilitates a highly specific multi-parameter cell staining for flow sorting as well as subsequent complete release of the conjugate probes from the sample. These new conjugates rely on recombinantly engineered antibody fragments that individually possess low epitope binding affinities. A tailor-made covalent conjugation chemistry allows for their multimerization as well as fluorochrome labeling, generating fluorescent probes comparable to conventional antibody conjugates. Importantly, upon addition of a release reagent, the antibody-fluorophore conjugates can be rapidly released from the cell surface. Accordingly, previously blocked epitopes are free for re-labeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. The versatility of this technology is presented in multi-parameter panels enabling clear distinction and discrimination of different cell populations. The applicability of the new REAlease® antibody fluorochrome conjugates is also demonstrated by flow sorting of human naïve regulatory T cells.
Comprehensive Benchmarking of T-cell epitope prediction tools
Authors: Paul Sinu , Peters Bjoern 
Published: January 2023

Abstract
Computational prediction tools are commonly used to select T cell epitope candidates in several immunological applications including vaccine design, cancer neoantigen identification, development of diagnostics and removal of unwanted immune responses against protein therapeutics. Most of these prediction tools are based on machine learning algorithms trained on MHC binding data or naturally processed MHC ligand data. The ability of currently available tools to predict T cell epitopes has not been comprehensively evaluated. In this study, we use a recently published dataset that systematically defined T cell epitopes recognized in vaccinia virus infected mice to evaluate the performance of all currently publicly available computational T cell epitope prediction tools. Using the same data set across all prediction methods provides an unbiased performance evaluation. The derived performance metrics allow immunologists to rationally decide on which prediction method to apply for their work at hand.
Development of PureQuant Real-Time PCR-Based Assay for Assessment of Immune Cell Identity and Purity in CAR-T Workflows
Authors: pradhan suman , Guzman Jerry , Landon Mark , Dargitz Carl , Lakshmipathy Uma 
Published: January 2023

Abstract
Significant progress has been made in harnessing the power of immune system, in particular, T cells, to treat certain kinds of cancers. One of the key challenges in developing immune cells as therapeutic agents is the accurate estimation of their identity and purity. Current methods used for the characterization of immune cell types rely on flow cytometry. Flow cytometry can accurately estimate CD8+ T lymphocytes and other surface markers, but the method is challenging to implement in a GMP manufacturing environment posing logistical challenges such as requirement for live cells, variability leading to difficult in standardizing and high throughput. In addition, cytometric methods are not accurate for specific intracellular targets that positively identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Therefore, there is an emerging need for alternative assay methods. Methylation state is known to be unique for specific cell types and can thus be used as an identifier in heterogeneous population of cells. Exploiting differences in cell type-specific methylation signatures, we developed assay kits that quantify the percentage of Treg and Th17 by detecting methylation status of FoxP3 and IL17A via qPCR of bisulfite converted genomic DNA. In contrast to flow analysis, sample requirement is minimal and the assay works well with fresh/frozen cells or genomic DNA. This assay has been implemented to accurately identify and estimate different T cell population in Chimeric Antigen Receptor (CAR)-modified T cells. The combination of accuracy, low sample requirement and flexibility provides an ideal measurement system for confirmation of identify and purity of T cell types critical for therapeutic applications.
Buoyant microbubbles as alternatives or adjuncts to magnetic bead methods for high-purity cell isolation
Authors: Younger John G , Roussey Jon , Bao Jianjun , Co Nadia Petlakh , Jones Thomas , Ostruszka Leo 
Published: January 2023

Abstract
Widely used in immunologic applications, magnetic bead systems have significant limitations including cumbersome workflows, generation of high-shear and high-compressive forces during routine use, and limited performance in low and high volume samples. Buoyancy-activated cell sorting (BACS) overcomes these issues by using low-density microbubbles in lieu of magnets and iron beads to perform isolation. Here we report methods for three common uses: removal of extraneous RBCs prior to FACS, isolation of high-purity B lymphocytes from murine spleens, and improvement of a magnetic NK separation system by jointly applying BACS to remove contaminating NKT cells. RBC removal from leukopheresis-derived PBMC was performed with anti-CD235a-conjugated microbubbles. B cell isolation was performed with streptavidin microbubbles and a negative selection cocktail of biotinylated anti-murine antibodies. NK cell isolation was performed on PBMC using Miltenyi human CD56 MicroBeads and an additional purification step using streptavidin microbubbles and an anti-CD3 antibody. Each experiment used Akadeum microbubbles and a one-minute trituration to combine microbubbles with cell suspensions. RBC microbubbles provided 98.3 ± 1.0% removal of RBCs with 11.3 ± 0.9% nonspecific binding. B cells isolated with microbubbles were 91.8 ± 2.8% pure with a yield of 94.2 ± 4.3%. NK purification with magnetic microparticles produced 80.5 ± 0.7% purity. Additional purification with microbubbles increased purity to 90.8 ± 1.3%. Microbubbles are a flexible and simpler alternative to magnetic cell isolation systems, delivering comparable performance and, when used in conjunction with magnetic beads, superior purity.
Physical cognate pairing of mouseTCR alpha-beta VDJ-rearrangements with phenotype directly from FACS sorted cells
Authors: Brown Brittany E , Byrne-Steele Miranda , Pan Wenjing , Hou Xiaohong , Depinet Mollye , Eisenhower Mary , Han Jian 
Published: January 2023

Abstract
Phenotypic characterization of single cells paired with immune receptor information can provide valuable insight into T cell function, which may be beneficial for the development of personalized therapies. In order to comprehensively analyze T cell function and specificity, it is necessary to integrate antigen receptor sequencing with phenotypic markers at the single cell level. We therefore developed a sensitive technology called iPair+™ that enables the simultaneous amplification and sequencing of paired mouse TCR alpha and beta chains with functional gene markers in a single, sorted immune cell via a combination of flow cytometry, multiplex PCR, and next generation sequencing. We utilized iPair+™ to study CD4 and CD8 T cells isolated from treated and non-treated mice from a breast cancer model. CDR3 diversity, clonality, and frequency were analyzed together with 80+ phenotypic genes. When cells isolated from both treated and non-treated mice were compared, we found gene expression levels changed significantly with treatment. Our data demonstrates that genes that were upregulated prior to treatment were associated with cancer growth, while genes upregulated after treatment were associated with T cell activation and function in cancer repression or growth inhibition. Our study indicates that iPair+ technology could reveal important insights into T cell function, clonal development, and immunotherapy response during treatment.
Investigating the human immune response with Myriad RBM’s TruCulture system and the NanoString nCounter Autoimmune Profiling Panel
Authors: Elliott Nathan A , Hwang Shen-An , Bailey Michael , Bailey Christina , LaBrie Samuel , Warren Sarah 
Published: January 2023

Abstract
TruCulture is a whole blood collection and culturing system designed for in vitro modeling of human immune responses. TruCulture retains all circulating immune cells, soluble factors, and Fc receptor expressing cells within a closed culture environment. The NanoString nCounter gene expression platform enables direct, enzyme-free counting of mRNA molecules from samples such as blood and biofluids with a total assay time to results around 24 hours. The Human Autoimmune Profiling Panel assays 770 genes (35 unique pathways) involved in autoimmunity. Additionally, the panel includes a cell profiling feature for measuring the relative abundance of immune cell types as well as gene targets for over 30 approved and investigational therapies. Whole blood from healthy donors is collected and exposed to potent immunostimulatory agents (SEB or TNF-a) in TruCulture tubes. Cell pellets are collected for total RNA isolation and nCounter gene expression analysis. In parallel, culture supernatants are analyzed for secreted cytokines using the Myriad OptiMap profiling system. Unsupervised hierarchical analysis of transcriptional profiles differentiates blood samples based on treatment groups. Both single gene and pathway level analysis identified significant differences between the treatment groups. Replicate TruCulture blood tubes produce gene expression data with high technical reproducibility (R2 &gt; 0.96). The data also demonstrates excellent correlation of RNA targets and protein abundance for key cytokines from the same blood sample. These results indicate the feasibility of combining TruCulture blood culture with NanoString gene expression analysis for the study of relevant pathological conditions from clinical samples.
Microchip platform for imaging-based efficacy testing of cells and reagents for immunotherapy
Authors: Onfelt Bjorn , Guldevall Karolin , Brandt Ludwig , Olofsson Karl , Carannante Valentina , Sandström Niklas , Wiklund Martin 
Published: January 2023

Abstract
Driven by a wave of new and improved methods for single cell analysis, e.g. flow cytometry, mass cytometry and single cell sequencing, it is increasingly recognized that there is a vast heterogeneity between individual cells within traditional cell classes. However, most of these techniques give a static picture of the molecular content in individual cells and provides limited information about plasticity or how the molecular content is translated into function, and it is easy to miss rare cell populations with functions important for mediating clinical effects. Current experimental approaches require large amounts of material, often impossible to obtain in clinical settings. Therefore, techniques that allow detailed functional evaluation of immune cells with single cell resolution from small samples are critically needed to advance assessment of therapies. We have developed a microchip platform for assessing cellular dynamics and function, with focus on assays for determining heterogeneity in cytotoxic response between individual NK or T cell. This microchip approach allows detailed characterization of thousands of individual cells within larger populations. By mounting an ultrasonic transducer on the chip we can also generate tumor spheroids that serve as model systems for studies of how NK and T cells attack solid tumors and respond to changes in the tumor microenvironment. Here I present this platform and give a few examples of how it has been utilized to generate mechanistic understanding of the cytotoxic process, classify cells based on response and to test efficacy of drugs for immuno-oncology and cells expanded for therapy.
Mapping leucocyte populations in mouse lymphoid tissues and blood using a 28-color panel on the BD FACSymphonyTM System
Authors: Baracho Gisele , Widmann Stephanie , Sisouvanthong Chad , Tyznik Aaron , Saksena Suraj 
Published: January 2023

Abstract
Multiparameter flow cytometric analysis of single cells allows the enumeration and characterization of heterogeneous cell populations in a variety of infections, blood cancers, immunodeficiencies and other disorders. Supported by advances in reagent development and instrumentation, this approach has increased dimensionality in single-cell data collection, and accelerated the discovery and testing of new therapies. Here we show a broad immunophenotyping panel that identifies the major leucocyte populations and various sub-populations from different mouse tissues. The panel can also be divided into focused modules for specific analysis of T cell, B cell or myeloid/natural killer cell lineages. BD FACSymphony combined with BD Horizon Brilliant Dyes allowed for high resolution identification of known leucocyte populations. Dimensionality reduction with t-SNE and automated clustering algorithms simplified cell analysis, enabling a faster and more efficient comparison of cell phenotypes across the tissues. Importantly, automated cell clustering analyses, while recognizing the predicted cell populations, also identified cell subsets missed during supervised analysis of the dataset. Thus, we describe a comprehensive single-cell profiling application capable of identifying well-characterized leucocyte populations and that has the potential to discover unique cell phenotypes.
Lipid composition impacts the assembly of MHC class II molecules in nanodiscs
Authors: Becker Tynan A , Osan Jaspreet , Rivera Kristian , Polanco Jonilee , Ferrante Andrea , Kuhn Thomas 
Published: January 2023

Abstract
Class II major histocompatibility complex molecules (MHCII) are transmembrane glycoproteins that display peptides generated and selected by intracellular antigen processing and presentation mechanisms to CD4+T cells, initiating an adaptive immune response. MHCII is expressed on the cell surface and in endosomal compartments, each of which have their own signature membrane characteristics. The traditional strategy to investigate peptide binding to MHCII has relied on the use of soluble MHCII, in which the transmembrane portion has been removed. This work studied MHCII in its native, membrane-embedded form. To this end, we used nanodisc, a synthetic model membrane device, to evaluate the effect of membrane lipid composition on MHCII assembly. The full-length human MHCII allele HLA-DR1 (DR1) was isolated from B-lymphoblastoid cell lines via immunoaffinity chromatography. Three types of nanodisc were generated: simple, fluid disordered and rigid ordered, each of them characterized by a unique lipid composition. Nanodiscs were separated using fast protein liquid chromatography (FPLC), the readings from which showed clear differences between nanodisc types. We observed that the fluid disordered nanodisc showed DR1 assembly as a cluster, with one major FPLC-generated peak. In contrast, simple and rigid nanodiscs were distinguished by multiple peaks. As DR1 tends to form aggregates, we inferred that in simple and rigid nanodiscs DR1 tetramers and dimers form in separate nanodisc assemblies. Taken together, our results indicate that membrane lipid composition has a strong impact on native MHCII assembly. As such, we predict that MHCII conformation and activity are function of the cell compartment they reside at any point in time.
Immunomagnetic cell separation is a suitable method for T cell electrophysiology and ion channel pharmacology
Authors: Tajti Gabor , Szanto Tibor G. , Racz Greta , Csoti Agota , Hajdu Peter , Panyi Gyorgy 
Published: January 2023

Abstract
Immunomagnetic cell separation is a powerful tool of immunology, however the presence of beads on the cell surface might alter the electrophysiological and pharmacological parameters of the ion channels expressed in the separated cells. We addressed this issue by measuring the aforementioned properties of Kv1.3, the predominant voltage-gated K+ channel of activated T cells upon immunomagnetic separation.
PBMCs from healthy donors were activated then CD4+ cells were separated with REAlease® CD4 Kit (Miltenyi Biotec Inc.). As control, we made stepwise removal of REAlease® antibody-bead complex, immunomagnetic negative separation and FACS. Purity and viability was determined by flow cytometry. Electrophysiology studies were made with patch-clamp technique in whole-cell configuration.
The presence of bead-antibody complex statistically significantly increased the time constant (τact) characterizing the activation kinetics of the current as compared to negative selection (p=0.049) and antibody only (p=0.024) controls, however the increase in τact from ~0.5 ms to ~0.6 ms does not bear any biological significance. Other gating parameters of Kv1.3, such as inactivation time constant and midpoint of voltage-dependence of steady-state activation were unaffected by the beads. Moreover, the binding of a peptide (ChTx) and a small organic molecule (TEA) inhibitor to Kv1.3 was oblivious to the presence of beads. In addition, the electrical capacitance of the cell membrane was not altered when beads were attached to the cells.
Based on our results we conclude that the application of immunomagnetic cell separation is completely suitable to electrophysiology studies of immune cells.
Lupus mice treated with Lupresan decreased auto-antibodies titers and improve facial lesions
Authors: Baeza Carlos Wong , Mora Albany Reséndiz , Pensado Monserrat Figueroa , Patiño Sandra Vela , Estrada Griselda Nava , Ramírez Carlos Wong , Ramírez Isabel Baeza 
Published: January 2023

Abstract
The nonbilayer phospholipid arrangements (NPA) are lipid associations different from the bilayer that are transient, so they are not immunogenic. But if they are stabilized by some drugs such as chlorpromazine, they become immunogenic and induce anti-NPA antibodies, which are found in patients with Lupus and are the ones that trigger a disease similar to Lupus in mice. Chloroquine, a drug used in the treatment of malaria, has been widely used to treat patients with Lupus; however, the mechanism of action by which it works in the treatment of Lupus is not known with certainty. In our research group, we have shown that chloroquine prevents the formation of NPA or reverts the formed, so it could be its mechanism in the treatment of Lupus. On the other hand, we have also shown that the polyamine spermidine is more effective than chloroquine in preventing the formation of NPA or in reverting the formed ones. Probably because this polyamine better stabilizes the lipid bilayer, because it has 3 positive charges in its structure at physiological pH, unlike the chloroquine that only has two. However, although polyamines participate in many biological functions, they cannot be used as drugs because when they have been administered in humans, they produce hematuria, proteinuria, renal failure and nephrotoxicity. In addition, in laboratory animals, they produce seizures, coma and death. So, in this work we design and synthesize Lupresan, an analogue of chloroquine with three positive charges at physiological pH. Lupresan was more effective in reverting or preventing the formation of NPA than chloroquine or spermidine, and as a result, it decreased auto-antibodies titers and healed the malar rash in mice with lupus to a greater extent than chloroquine.
Improved antimalarial delivery to inflammatory cells driving lupus pathogenesis using shape-based targeting nanoparticles
Authors: Allen Marilyn , Karabin Nicholas , Golding Amit , Scott Evan , Szeto Gregory 
Published: January 2023

Abstract
Systemic lupus erythematosus (SLE) is a chronic disease that attacks healthy organs via self antigen-autoantibody immune complexes. These complexes activate plasmacytoid dendritic cells (pDCs) via (toll-like receptor) TLR7 and 9, driving inflammatory type I interferon (IFN). Antimalarials, hydroxychloroquine (HCQ) and chloroquine (CQ), are TLR antagonists used to alleviate inflammation in SLE patients, but require ~3+ months before therapeutic efficacy. Recent work reports 7.5% patients treated with HCQ are at risk of irreversible retinopathy after 5 years, which increases to 1 in 5 patients with duration of therapy. We hypothesized that PEG-b-PPS filomicelles (FMs) will improve drug activity and reduce toxicity by directly delivering HCQ or CQ to pDCs via passive, shape-based targeting. Human peripheral blood mononuclear cells (hPBMCs) were treated with soluble CQ or CQ in FMs, stimulated with CpG-A ODN 2216 and quantified for type I IFN via myxovirus-resistance 1 (MX1) gene expression using real-time RT-PCR. Our results show that 50 μg CQ/mg FM decreased MX1 expression 20 fold to soluble CQ and 10 fold to CpG-A ODN 2216 control, while CQ in control polymer PLGA spheres had no effect. Additionally, cellular-level distribution of FMs in hPBMCs show that FMs accumulate in pDCs more than B and T cells over a 48 hour time course. These results show polymer shape impacts drug delivery, CQ-FMs inhibit SLE-relevant pathways and CQ-FMs may be effective and more targeted than CQ.