bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dominant interfering CARD11 variants disrupt JNK signaling in T cells
Authors: Bauman Bradly M. , Chan Alice Y. , Snow Andrew L. 
Published: February 2023

Abstract
Previous studies in mice implicated c-Jun N-terminal kinase 1 and 2 (JNK1/2) as important determinants of helper CD4+ T cell differentiation through their influence on AP-1 and NF-AT-dependent gene transcription. However, mechanisms governing the JNK signaling pathway in human lymphocytes remain nebulous. CARD11 is a critical scaffold protein in T and B cells that connects antigen receptor (AgR) engagement to downstream transcriptional programs responsible for effector cell survival, proliferation and differentiation. Although CARD11 is best known for facilitating AgR-induced NF-κB activation, a handful of studies have shown it is also required for AgR-induced mTORC1 and JNK2 signaling. Using both CARD11-deficient Jurkat T cells and primary human T cells, we found that phosphorylation of both JNK1 and JNK2 in response to T cell receptor (TCR)/CD28 activation is dependent on CARD11. Furthermore, we demonstrate that heterozygous dominant negative (DN) CARD11 mutations derived from patients with CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease also disrupt TCR-dependent JNK1/2 phosphorylation and c-Jun accumulation with variable potency, closely mirroring the extent of impaired NF-κB activation. Considering that loss of either JNK1 or JNK2 in mice contributes to reduced IFN-γ production and skewed Th2 phenotypes, our findings suggest that defective JNK signaling in CD4+ T cells may contribute to severe atopic disease manifestations noted in most CADINS patients. Ongoing studies will define differences in specific JNK-dependent gene signatures that correlate with specific CARD11 DN variants and clinical phenotypes.
Intravital three-photon microscopy of T-cell migration in the T-cell zones over the entire depth of mouse popliteal lymph node.
Authors: Choe Kibaek , Hontani Yusaku , Wang Tianyu , Hebert Eric , Ouzounov Dimitre , Xu Chris 
Published: February 2023

Abstract
Most T cells in a popliteal lymph node are located in T-cell zones at ~150 to ~600 μm below the cortical surface of the lymph node. For the last 20 years, intravital two-photon microscopy has been a powerful tool for observing the three dimensional T-cell migration in the T-cell zones. However, two-photon imaging can only visualize superficial T-cell zones to ~300 μm depth. In this work, we successfully visualized T-cell migration in T-cell zones over the entire depth of a mouse popliteal lymph node with intravital three-photon microscopy. We measured the change in lymphocyte velocity with increasing laser powers at 600 μm depth to determine the safe laser power range for visualizing T-cell migration. We found that, with less than 80 mW at 1300 nm excitation, the migration of EGFP-expressing lymphocytes were not perturbed by the laser illumination. Using the safe power of 1300 nm excitation, we observed dynamic migration of EGFP-expressing naïve CD4 T cells and DsRed-expressing naïve CD8 T cells simultaneously in T-cell zones at various depths. The naïve T cells were sparse at 0 ~150 μm depth in cortical side (follicular and interfollicular regions), dense in the T-cell zones, and less dense in the medulla, which was identified by labeling with eFluor615-conjugated LYVE-1 antibody in vivo. We found that the density distributions of CD4 and CD8 T cells were different in the T-cell zones. We also measured the migration velocity and motility coefficient for naïve CD4 and CD8 T cells in the T-cell zones over the entire depth of the lymph node. This work shows that three-photon microscopy has the potential to shed light on immune cell dynamics in the deep regions of lymph nodes that are typically inaccessible to conventional two-photon microscopy.
EasySep&[trade] T75 Magnet: A Novel Magnetic Platform for Large-Volume Cell Isolation from Whole Blood and Leukapheresis Packs
Authors: MacDonald G. Neil , Toombs Eric , de Jong Susan , McQueen Karina L , Eaves Allen C , Louis Sharon A. , Kokaji Andy I 
Published: February 2023

Abstract
Isolating cells from large volume-samples such as leukapheresis or whole blood bags is a common procedure that precedes many immunological studies. Working with large volumes can be challenging as the sample often needs to be split and processed in parallel, which can be tedious and may delay downstream studies. The EasySep™ T75 Magnet simplifies cell separation procedures when processing up to 225 mL of leukapheresis sample or 125 mL of whole blood, and is based on column-free immunomagnetic EasySep™ cell isolation technology.
Negative selection protocols have been optimized for isolation of human T cells (purity: 95.3 ± 2.5%, n=5), CD4+ T cells (purity: 97.2 ± 1.4%, n=4), CD8+ T cells (purity: 91.9 ± 2.2%, n=5), and monocytes (purity: 91.5 ± 1.0%, n=3). Positive selection protocols have been optimized for isolation of CD3+ cells (purity: 95.4 ± 2.9%, n=9), CD4+ cells (purity: 88.8 ± 3.2%, n=3), CD8+ cells (purity: 95.1 ± 2.4%, n=6), and CD14+ cells (purity: 96.2 ± 0.7%, n=3). Protocols for depletion of red blood cells (RBCs) (residual RBCs: 2.5 ± 1.7%, n=5) and enrichment of peripheral blood mononuclear cells from whole blood (purity: 99.2 ± 0.5%, n=5) have also been developed.
Notably, these protocols can be completed in 20 – 26 minutes, making the EasySep™ T75 Magnet the fastest and simplest method for isolating highly purified immune cells from large-volume whole blood and leukapheresis samples.
Leveraging high-dimensional flow cytometry to reveal the human immune system at a population-wide scale
Authors: Liechti Thomas Andreas , Beddall Margaret , Van Gassen Sofie , Novák David , Ballard Reid , Iftikhar Yaser , Mangino Massimo , Saeys Yvan , Spector Tim , Roederer Mario 
Published: February 2023

Abstract
Population-wide assessment of the human immune system is needed to better understand immune homeostasis, immune responses against pathogens or the efficacy of vaccines. High-dimensional flow cytometry is the gold standard for in-depth immunophenotyping but the assessment of large cohorts requires multiple experiments over a prolonged period of time and bear multiple sources of error, reducing precision required for sensitive analyses. We performed a large immunophenotyping study using 28-color flow cytometry to investigate the heritability and homeostasis of the immune system and T cell functions in 3000 individuals. Here, we describe our high-throughput sample and data processing pipeline (200 samples/day) in combination with high dimensional flow cytometry to dramatically reduce the number of experiment-days in our study, resulting in low variation and high reproducibility. We demonstrate several carefully optimized strategies to reduce experimental errors and inter-assay variation at every level. In addition, we discuss strategies for pre-processing and analysis of large flow cytometry datasets using manual gating and unsupervised clustering analyses, such as FlowSOM, in view of the fact that most current strategies are highly limited for large datasets.
In conclusion, we established a high-throughput high-dimensional flow cytometry pipeline enabling the analysis of thousands of samples with low technical and experimental variation. Our pipeline in combination with our three 28-color flow cytometry panels is adaptable to other large cohort studies for which precision and accuracy over time are important.
ctDNA technology for early cancer detection in the Pacific setting – the importance of Talanoa
Authors: Sika-Paotonu Dianne , Walton Mischa , Faavae Sepola , Guilford Parry 
Published: February 2023

Abstract
For Pacific Peoples living in Aotearoa/New Zealand, the burden of cancer remains a serious health concern. A promising early cancer detection approach involves the use of circulating tumour DNA (ctDNA) found in the blood circulation. Current ctDNA approaches focus on the analysis of ctDNA derived from established tumours in patients, to help provide information relating to the cancer’s size and progression using specific laboratory isolation and quantification techniques. Adaptation of these current ctDNA technologies used in the cancer monitoring setting, for early diagnosis, would generate a cancer detection approach easily initiated by obtaining ctDNA from DNA fragments found within a blood sample. To understand the potential utility of ctDNA technology for early cancer detection within the Pacific setting in Aotearoa/New Zealand, engagement was sought with Pacific leaders using Talanoa, focused on cancer and early cancer detection. Interim analyses highlighted factors considered directly relevant to Pacific Communities and the cancer setting including the need for health technologies and systems that align with cultural contexts for Pacific Peoples and their families. Support was also expressed for the development of an early cancer detection tool utilising ctDNA as a simple methodological approach, with acknowledgement of the potential for this method to address current barriers to cancer care and services. This work highlights the importance of ensuring cultural context is understood and respected when engaging with Pacific Communities, and also within the cancer and health care setting.
Micro-structure Proteins Characterization in Tissue Sections and Clinical Biopsy Samples by a Novel Spatial Proteomic Method Named AutoSTOMP
Authors: Yin Bocheng , Ewald Sarah , Holmes Jeff , McGowan Emily 
Published: February 2023

Abstract
We aim at structure determining immunological machinery using a novel spatial selective proteomic tool, autoSTOMP, developed in Ewald lab. AutoSTOMP comprises of UV initiated biotin tagging by confocal microscopy, affinity isolation, and peptide identification by the mass spectrometer, rendering a precise characterization of micro-structures of interest (mSOIs) at intracellular organelle scale excluding surrounding area with the decent signal-to-noise ratio. Previously, autoSTOMP was employed to investigate the parasitophorous vacuole membrane, a unique intracellular membrane structure in apicomplexan infected cells. We broaden the application of autoSTOMP to respond to the calls from the research or clinical laboratory for a reliable, repeatable, and re-constructible protocol to explore the mSOIs of an animal specimen or a patient biopsy. We demonstrated, in the cardiac infarcted tissue in ischemic injured rat, the mSOIs stained against the macrophage/monocyte marker, CD68, are enriched in lysosomal proteins, and characteristic proteins involved in the Fcγ receptor-activated macrophage deformation, migration, and phagocytosis. Furthermore, in eosinophilic esophagitis (EoE) biopsy, we identify the lgG4+ cell-cluster structures are enriched in eosinophil-derived granule proteins and lgE receptor FcɛRI signaling associated proteins as well as food allergens to build up the correlation between patient’s diet with EoE progression.
Multiomic characterization of T-cell populations at the single-cell level utilizing sensitive dextramers and BD® AbSeq on the BD Rhapsody™ Single-Cell Analysis system
Authors: Lopez-Salmeron Vadir , Sakofsky Cynthia , Jacobsen Kivin , Nakamoto Margaret , Brix Liselotte , Mortimer Stefanie 
Published: February 2023

Abstract
Adoptively transferred antigen-specific T cells have shown great efficacy in treatment of some virus-associated diseases and malignancies. A major driver of the development of adoptive T-cell therapy has been our ability to successfully characterize the functional status and antigen specificity of T cells. However, this has been limited by inefficient detection of antigen-specific T cells possibly due to their low frequency and low binding affinities to known MHC-peptide complexes. Here, we aim to combine two powerful technologies, advanced dCODE™ Dextramer® from Immudex and single-cell multiomics analysis using the BD Rhapsody™ Single-Cell Analysis system, to detect and characterize disease-specific CD8+ T cells within thousands of PBMCs. Currently, we are able to identify over 350 mRNAs alongside a panel of over 20 BD® AbSeq cell surface protein markers which can be associated with T cell activation states. These data can be used to define T-cell phenotypes alongside antigen specificity of enriched CD8+ dextramer+ cells from a PBMC population. This study outlines our ability for high-resolution T-cell profiling that has broader implications and utility in immuno-oncology, infectious diseases and autoimmunity.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.
A single-tube, 44-marker CyTOF assay to assess antigen-specific immunity in whole blood human samples with data analysis solution
Authors: Mujib Shariq , Li Stephen , Zabinyakov Nick , Loh Christina 
Published: February 2023

Abstract
Monitoring the immune response in the setting of infectious disease and cancer is critical to assess disease status and targets of immune therapy. CyTOF® mass cytometry enables multiplex cellular phenotyping with more than 50 markers, making it ideal for comprehensive immune profiling. CyTOF technology utilizes antibodies tagged with unique monoisotopic metals, resulting in distinct signals that provide a high-resolution multiparametric landscape of a single cell. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, 30-marker antibody cocktail for immune profiling of human whole blood and PBMC by CyTOF. Paired with Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations including major lineages and their subsets. In this study, we expanded the 30-marker assay to a 44-marker panel including exhaustion markers such as PD-1 and CTLA-4, co-stimulation markers 4-1BB and ICOS, and intracellular cytoplasmic markers IFN-γ, TNF-α, IL-2, perforin and granzyme B to assess cellular function in PMA/ionomycin-stimulated whole blood cultures. We modified the existing Maxpar Pathsetter model to automate the analysis of the expanded panel and report on additional functional parameters such as T cell exhaustion and cytokine production. Next, we applied this panel to whole blood stimulated with CMV peptides to investigate antigen-specific immune responses in a viral infection model in concert with in-depth phenotypic assessment. Collectively, we demonstrate the flexibility of the Maxpar Direct Immune Profiling Assay to incorporate additional surface and intracellular markers to study antigen-specific immunity in the context of whole blood immune profiling.
For RUO.
Practical Cell Counting Method Selection to Increase the Quality of Cell Counting Results
Authors: Chan Leo , Qiu Jean 
Published: February 2023

Abstract
The importance of cell counting has increased significantly in the last decade due to the major advances in the fields of cell and gene therapy, biologics production, and regenerative medicine. This has necessitated the development of a standardized approach to cell counting assays. In the recent years, the U.S. Food and Drug Administration (FDA), in collaboration with the National Institute of Standards and Technology (NIST) and the International Organization for Standardization (ISO), has launched an effort to standardize cell counting methods to improve the confidence in cell counting measurements.
There is a wide range of biological sample types, various formulations, and bioprocessing steps for cell and gene therapy products. Furthermore, there are no ground truth reference materials for live cells makes determining the accuracy of cell counting difficult defined in the ICH Q2 (R1). Therefore, in order to increase the confidence of cell counting results, it is critical to follow the two recently published ISO cell counting standards to ensure cell counting methods are tailored to the specific sample and purpose.
In this work, we will provide insights and guidance for the ISO standards for method selection. The important aspects of the ISO cell counting standards have been distilled to six key factors for selecting the cell counting methods. Attention to these details will allow researchers to more easily adhere to the ISO recommendations to ensure high quality cell counting measurements, and in doing so, sites can meticulously document their rationale, process, and data results.
Development of a dried pre-formulated antibody panel (7 colors/17antibodies) for the identification of human innate lymphoid cells by flow cytometry
Authors: Lacaille Vashti , Miloud Tewfik 
Published: February 2023

Abstract
Innate lymphoid cells (ILC) play an important role in innate immunity, lymphoid organogenesis and tissue remodeling. They form a heterogeneous population that can be categorized into 3 groups (i.e. ILC1, ILC2 and ILC3) based on their phenotype and/or distinct patterns of cytokine production. The identification and enumeration of different blood ILC subsets is important to understand immune regulation in disease models such as allergy, cancer and inflammation. Flow Cytometry is the method of choice for analysis of ILCs in human blood. However, as ILC frequencies may range below 0.1% of white blood cells, considerably elevated sample volumes and/or cell-enrichment is required. Furthermore, due to the lack of specific markers for definition of ILCs, at least 15 antibodies are required for mere identification of ILCs, including a lineage-exclusion channel to exclude most of the leukocytes. Based on current knowledge in human ILC biology, we have composed a respective set of markers to design a dry unitized 17 antibodies/7-color panel (CD294/Lineage/CD117/Nkp46/CD127/CD161/CD45) for flow cytometry. The panel in dry DURAClone format allows for the reliable identification of all ILCs subsets in whole blood or peripheral blood mononuclear cell fractions (PBMCs) in concordance with current scientific opinion. Due to the elimination of the pipetting of 17 antibodies, the method can substantially reduce variability and error related to sample preparation. Furthermore, the streamlined workflow can facilitate inter-laboratory transfer of the assays in multi-site studies.
Targeting SLP76:ITK interaction separates GVHD from GVL in allo-HSCT
Authors: Karimi Mobin A , Mammadli Mahinbanu Mahinbanu , Huang Weishan , Harris Rebecca , Weeks Samuel , May Adriana , Gentile Teresa , Henty Ridilla Jessica L. , August Avery , Karimi Mobin 
Published: February 2023

Abstract
Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for relapsed hematological malignancies due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also primarily mediated by the same donor T cells. Here we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in T cell-mediated GVL vs. GVHD effects after allo-HSCT. Neither CD8+ T cells nor CD4+ donor T cells from mice expressing a tyrosine to phenylalanine mutation at position 145 (Y145F) of the adapter protein SLP-76 caused GVHD, T cells cells preserved GVL effects after allogeneic transplantation. SLP76Y145FKI CD8+ and CD4+ donor T cells also produce less inflammatory cytokines and show decreased migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). We also report the development of a novel peptide that can specifically inhibit SLP76 and ITK interactions, which results in decreased phosphorylation of PLCγ1 and ERK, and decreased cytokine production in human T cells. This peptide inhibited donor T cell-mediated GVHD while maintaining GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to reduce GVHD while retaining the beneficial GVL effects after allo-HSCT treatment.
TCF-1 acts as a positive regulator in alloactivated T cells, and TCF-1 deficiency reduces GVHD yet maintains GVT responses
Authors: Harris Rebecca , Karimi Mobin A 
Published: February 2023

Abstract
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for hematological malignancies. Mature donor T cells in this graft help kill residual malignant cells (graft-versus-tumor or GVT effect). However, donor T cells also damage healthy host tissue, causing graft-versus-host disease (GVHD) in about 30–70% of allo-HSCT patients. These alloactivated T cells proliferate, migrate to target organs (liver, skin, and small intestine), and produce cytokines during GVHD. Our work focuses on modulating T cell signaling to separate GVHD from GVT, as GVHD is the main clinical barrier to widespread use of allo-HSCT.
T Cell Factor-1 (TCF-1) is critical for T cell development and activation, but its role in alloactivated and mature T cells is unclear. Using a mouse model of MHC-mismatched allo-HSCT leading to GVHD, we examined the role of TCF-1 in this context. Loss of TCF-1 specifically in T cells led to reduced severity and persistence of GVHD, and reduced host tissue damage from both CD4 and CD8 T cells, yet maintained GVT effects. Cytokine production, proliferation, and survival of donor T cells, as well as donor T cell phenotype, were all affected by loss of TCF-1. Therefore, TCF-1 controls mature T cell phenotype and function during allo-HSCT, and is indispensable for GVHD damage. TCF-1 is dispensable for GVT effects, because loss of this factor does not disrupt antitumor immunity. Thus, loss of TCF-1 separates detrimental GVHD damage from beneficial GVT effects.
This work will help to improve outcomes for allo-HSCT patients, who have poor survival due to GVHD and increased risk of deadly infections. This research also enhances our basic understanding of how alloactivated mature T cells are regulated during allo-HSCT.
IL-33 is a critical early activation and differentiation signal for alloreactive CD4+ T cells that enables their infiltration into GVHD target tissues
Authors: Dwyer Gaelen K , Mathews Lisa , Lucas Anna , Blazar Bruce R , Shlomchik Warren , Turnquist Heth Roderick 
Published: February 2023

Abstract
Graft vs host disease (GVHD) is a lethal complication after allogeneic stem cell transplantation (alloSCT), where donor T cells destroy host tissues. To create space for donor cells and prevent their rejection, alloSCT protocols use recipient conditioning that augments the stromal-derived cytokine interleukin (IL)-33 in the lymphoid organs (LO) and tissues. While we have established that IL-33 is a clinically-relevant signal in GVHD, mechanisms by which it contributes to alloimmunity are not well defined. Herein, we used IL-33 receptor, ST2, deficient B6 mice as T cell donors to elucidated IL-33-mediated mechanisms in the early activation and differentiation of alloreactive T cells. In our model, we transferred equal ratios of B6 Cd4-Cre x St2fl/fl (ST2ko) and Cd4-Cre (ST2wt) donor T cells into irradiated B6 and BALB/c recipients. Donor T cells in the LO and tissues were characterized at day 1–3, 5, and 7 post alloSCT. BALB/c recipients exhibited blunted early activation and proliferation of alloresponsive ST2ko CD4+ T cells compared to ST2wt CD4+ T cells in LO of the same recipient. ST2ko T cells in allogeneic recipients also failed to differentiation into Tbethi and CXCR3hi Th1 cells able to infiltrate into target tissues and cause GVHD. RNAseq analysis confirmed the need for IL-33-signaling for alloreactive Th1 generation as ST2KO CD4+ cells exhibited reduced IFNγ, Tbet, CXCR3, and instead expressed higher levels of Th2 and regulatory molecules (Foxp3, IL10, Gata3). IL-33 signaling was not required for the non-alloantigen driven lymphopenic expansion by CD4+ T cells in syngeneic recipients. These data establish a previously unappreciated and targetable role for IL-33 in effective alloAg-reactive donor Th1 generation after alloSCT.