bluuebunny/crossref_metadata_2025_split huggingface dataset:
3D extension of spatial patterning analysis of cellular ensembles (SPACE) to extract parameters for dynamic tumor modeling
Authors: Sreejithkumar Vivek , Schrom Edward , Germain Ronald 
Published: November 2024

Abstract
Investigating the cellular interactions within three-dimensional (3D) tumor microenvironments is essential for understanding tumor growth and progression. Building upon the success of our previous computational framework, SPACE, designed for spatial analysis of high-plex tissue images, we are developing 3D-SPACE. This innovation extends spatial pattern analysis to 3D volumetric tissue images. Volumetric images more accurately capture nuances of cellular arrangements and tissue structures, offering a more realistic representation of the tumor microenvironment.
Applying 3D-SPACE to analyze full 3D tumor datasets has the potential to reveal crucial parameters for understanding tumor outcomes. This approach not only elucidates previously undiscovered spatial patterns but also allows for the quantification of parameters such as contact probabilities between different cell types or the killing efficiencies of key effector cells in situ. These spatially-derived parameters can then be used in predictive equations that model and forecast tumor growth based on cellular interactions.
Using a 3D tumor model and 3D-SPACE, we can quantify cell interactions and develop predictive equations for tumor growth dynamics, with the aim of deriving a predictive reproduction number (R₀) for tumor growth. Working towards a predictive R₀ for tumor growth offers a valuable approach for predicting tumor growth outcomes.
This work was supported in part by the Intramural Research Program of NIAID, NIH.
Label-free morphological profiling and isolation of immune cell subsets using VisionSort, a novel, AI-based flow cytometry platform.
Authors: Schneider Greg , Wagatsuma Keisuke , Tsubouchi Asako , Packiasamy Juliet , Uematsu Mika , Nomaru Hiroko , Teranishi Kazuki , Akai Satoru , An Yuri , Komoriya Kaoru , Kawamura Yoko , Ota Sadao 
Published: November 2024

Abstract
Identification, characterization, and minimally invasive isolation of specific populations of human immune cells are critical for understanding and treating disease. Here we present data on label-free identification of three immune cell subsets by morphological profiling using VisionSort; a label-free, artificial intelligence (AI)-driven cellular analysis and sorting platform. By capturing single-cell digital phenotypes, we characterized mouse T-cells and generated ‘ground truth’ functional profiles for activated and non-activated T cells. A set of machine-learning derived classifiers was generated to identify these phenotypic classes in unlabeled T-cell subsets. The classifier showed an area under the curve (AUC) performance for differentiating between phenotypically defined T cell populations of 0.917. In addition, by using unsupervised machine learning, we were able to resolve activated and non-activated T cell populations label free, using morphological data alone. Using a similar approach, we show label-free differentiation/classification of B cells from plasma cells with an AUC score of 0.941 and M1 and M2 polarized macrophages with an AUC score of 0.878 +/- 0.002 (n=6). Here we report results on the use of a novel, label-free cytometry platform to characterize and isolate human immune cell subsets using morphological profiling and AI with applications for investigators in basic life sciences and drug developers in small molecule, antibody, and cell therapy R&D.
Single-cell full-length TCR/BCR profiling assay with high sensitivity and CITE-seq compatibility enables deeper immune cell characterization
Authors: Genero Gracia , Han Zhiyuan , Chen Lin , Gordon Chelsea , Vadrevu Samatha , Sakofsky Cynthia , Li Ruifang , Wang Larry , Padmanabhan Raghavendra , Jensen Devon , Ayer Aruna 
Published: November 2024

Abstract
Single-cell full-length TCR/BCR assays provide the ability to profile the highly diverse T cell receptor (TCR) and B cell receptor (BCR) repertoires of the adaptive immune response. The identification of T cell clonotype can be challenging due to insufficient sensitivity to detect low abundance TCR transcripts. Furthermore, cell type identification can be limited using only transcriptomic profiles.
Here, we present data with high resolution of TCR clonotype using a new highly sensitive TCR/BCR assay combined with CITE-seq using the BD™ AbSeq Assay. We profiled human and mouse T cells alongside whole transcriptome analyses with and without CITE-seq. The enhanced assay showed high clonotype detection with transcriptome-only profiling and with CITE-seq. Results also showed high gene expression correlation (R2&gt;0.97), consistent library quality and mapping, and better resolution of cell subtype clustering with CITE-seq. Compared to transcriptome-only profiling, the coupling of surface protein expression with transcriptomic and clonal information of each cell provides deeper insights of the immune repertoire and enhances detection of cell subsets within a complex population. The results demonstrate the utility of our improved immune profiling assay with highly sensitive TCR/BCR detection for in-depth multiomics analyses of single cells.
For Research Use Only. Not for use in diagnostic or therapeutic procedures. © 2023 BD. All rights reserved.
Direct cloning of novel and specific human neutralizing anti-interferon-alpha or anti-interferon-beta antibodies
Authors: Papasavvas Emmanouil , Lu Lily , Fair Matthew , Oliva Isabela , Cassel Joel , Majumdar Sonali , Mounzer Karam , Kostman Jay , Tebas Pablo , Bar-Or Amit , Muthumani Kar , Montaner Luis 
Published: November 2024

Abstract
Background: Type I interferons (IFNs) play a pivotal role in the development of immune responses and have significant clinical importance. However, dysregulated activation of type-I IFN response has been associated with a wide range of disorders. Therefore, it is crucial to develop tools that facilitate the understanding of their mechanism of action, enabling the development of more effective therapeutic strategies.
Methods: We identified, cloned, and characterized by enzyme-linked immunosorbent assay (ELISA), IFN receptor dimerization assay, signal transducer and activator of transcription 1 (STAT-1) phosphorylation, surface plasmon resonance, and western blot the binding/neutralizing/specificity properties of novel human anti-IFN-α and IFN-β monoclonal antibodies (Abs). The Abs were sourced from individuals with multiple sclerosis with a history or ongoing IFN-β therapy, and from antiretroviral therapy (ART)-suppressed individuals with chronic HIV-1 infection who had been treated with IFN-α.
Results: We observed that clones AH07856, and AH07857 effectively neutralize IFN-α-mediated IFN receptor dimerization and signaling but not IFN-β. Likewise, clones AH07859, and AH07866, neutralize IFN-β-mediated IFN receptor dimerization and signaling but not IFN-α.
Conclusion: We describe novel neutralizing Abs that selectively block human IFN-α or IFN-β with potential applications in research, and in therapeutics and other medical interventions.
Use of a Near Infrared Probe to Assess Intracellular Hydrogen Sulfide Production
Authors: Liu Jianyun , Mesfin Fikir , Zeng Lifan , Manohar Krishna , Hunter Chelsea , Shelley W , Brokaw John , Markel Troy 
Published: November 2024

Abstract
Hydrogen sulfide (H2S) is a ubiquitous gas and has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious, and neuropathological diseases have been undertaken. Numerous studies have shown that H2S can regulate PI3K/AKT, NF-κB and MAPK signaling pathways, suppressing LPS-induced inflammation. However, its use in the clinical setting and mechanistic studies are limited in part due to the lack of effective H2S detection methods. In the current study, we investigated a compound as a potential near infrared (NIR-HS) probe to measure intracellular H2S. After being synthesized and purified using chromatography, NIR-HS probe was added to a cell-free environments in the presence of hydrogen sulfide salts (Na2S and NaHS) or a H2S-slow-releasing donor (GYY4137) to determine its ability in detecting H2S. We further demonstrated that the NIR-HS probe can detect endogenous H2S in different cell lines. Detected by using the NIR-HS probe, knocking down and overexpressing the endogenous H2S synthases (CBS, CTH, and MPST) altered the production of H2S, respectively. Further studies are needed to demonstrate its use in large scale and in vivo environment to further demonstrate its effectiveness in detecting H2S.
Finding the needle in the haystack- detection of circulating endothelial cells with a new EpiSpot
Authors: Preyer Rosemarie , Strecker Kevin , Ettischer-Schmid Nicole 
Published: November 2024

Abstract
Revascularisation in the setting of critical limb ischema (CLI) is the treatment objective. For the evaluation of an angiogenic effect, peripheral circulating endothelial cells (CECs) and endothelial progenitor cells in the blood stream should be detected in an EpiSpot system. The development of the assay and proof of principle with human umbilical vein endothelial cells (HUVEC) are part of this work.
Therefore, vascular endothelial growth factor (VEGF), the coagulation factor VIII (FVIII), E-Selectin, von Willebrand Faktor (vWF) and Interleukin-6 (IL-6) antibody pairs were tested and concentration and incubation times were evaluated with HUVECs in combination with PBMCs. Flow-cytometric analysis was done in parallel to determine the applicability of HUVECs as a model for circulating endothelial cells. Beside this, isolated and enriched endothelial cells (ECs) from peripheral blood were characterised as CECs by using FACS.
E-Selectin has been selected to identify ECs unambiguously in the established assay system. This was proven by titration of HUVEC in PBMCs, and recovery rates of 10 HUVEC / 200.000 PBMCs were achieved.
The developed assay-system can detect low numbers of endothelial cells in general. The amount of circulating endothelial cells in healthy volunteers is relatively low, therefore additional experiments will reveal the usefulness of the assay for CLI treatments and will allow to define the sensitivity in patient samples.
Funding EU Horizon 2020(n° 733006 PACE)
Simultaneous profiling of single-cell BCR clonal diversity and whole transcriptomes from diseased and healthy PBMCs in a single experiment using split-pool combinatorial barcoding
Authors: Sapre Ajay , Papalexi Efthymia , Salvino Alec , Marrujo Simone , Gallareta-Olivares Guillermo , Roco Charles , Rosenberg Alexander 
Published: November 2024

Abstract
Many autoimmune diseases are characterized by aberrant B cells which play a central role in pathogenesis. Each B cell expresses a B cell receptor (BCR), which determines its specificity in binding to different epitopes. For autoimmune diseases such as Rheumatoid Arthritis, Lupus, and Type 1 Diabetes our current understanding of the role of unique BCR repertoires is poorly understood; however, recent advances in single-cell RNA sequencing has allowed the field to simultaneously profile both the BCR and whole transcriptome leading to the characterization of clonal diversity of B cell subtypes.
Traditional methods using microfluidic barcoding are low throughput (1,000s-10,000s of cells) and costly. To overcome these limitations, we developed a method to simultaneously characterize the BCR repertoire and full transcriptomes of up to 1 million cells from up to 96 samples in one experiment.
For proof of concept, we applied this method to simultaneously profile the BCR and whole transcriptome on multiple healthy and diseased samples. Using the whole transcriptome data, we detected all major subtypes of B cells in peripheral blood. From the BCR data, we defined BCR isotypes, captured a majority of cells with a paired chain, detected full length chains, and differentiated dominant & rare clones. This technology paves the way for high-throughput and sensitive screening of BCR repertoires to further the understanding of B cell biology and disease.
Defining healthy and breast tumor-associated neutrophils at a whole-organism, single-cell resolution
Authors: Hsu Alex , Alshetaiwi Hamad , Gong Yanwen 
Published: November 2024

Abstract
Neutrophils accumulate systemically in tumor-bearing organisms, and have been shown to promote primary tumor progression and metastasis through suppression of the anti-tumor immune response. However, neutrophils have specialized phenotypes in different organs, and their full transcriptional heterogeneity and maturation trajectory has yet to be defined across healthy and cancer states. Here, we performed single-cell RNA sequencing on isolated neutrophils from bone marrow, spleen, lymph nodes, blood, mammary fat pad/tumor, and lungs from wild-type and breast tumor-bearing MMTV-PyMT mice. Our analysis reveals distinct transcriptional programs which distinguish systemic neutrophil function in normal homeostasis and cancer. In wild-type mice, we describe a neutrophil maturation trajectory that begins with granulopoiesis in the bone marrow, continues development in the spleen and blood, and reaches a fully mature state in the lymph nodes, mammary fat pad, and lungs. This is captured by five maturation stages, N0-N4, each with its own characteristic gene signature. In MMTV-PyMT mice, we observe a systemic upregulation of genes including Ifitm1, Wfdc17, Clec4e, Igfbp6, and Saac, which have been implicated in cancer-induced immunosuppression. Taken altogether, this work provides a multi-organ perspective into neutrophil development across healthy and cancer states and identifies markers specific to breast tumor-associated neutrophils which may be potential immunotherapeutic targets.
IL3-driven crosstalk of T cell and basophil enhances antitumor immunity
Authors: Mayberry Colleen , Wei Jian , Chang Chih-Hao 
Published: November 2024

Abstract
Cytotoxic T lymphocytes (CTLs) are vital to combat cancer yet become exhausted in the immunosuppressive tumor microenvironment. Here we probed the role of CTL-derived IL-3 in enhancing CTL anti-tumor response and preventing exhaustion. Transwell culture of CTLs, genetic manipulation of T-cell IL-3 expression, and T cell and Il-4 deficient mice were used to evaluate factors in T-cell exhaustion, proliferation, and survival. We found reduced IL-3 production in intratumoral CTLs, correlating with exhaustion, and revealed that CTL-derived IL-3 influences basophils, synergistically amplifying CTL anti-tumor immunity. Introducing IL-3-activated basophils slows tumor growth and basophil depletion lessens IL-3 efficacy. Ingenuity Pathway Analysis of transcriptomic data revealed that IL-3-treated basophil-conditioned medium enhances T-cell function via IL-4 stimulation, resulting in altered cytokine signaling, inflammation, and T-cell activation. Augmenting IL-3 confers increased CTL activity, reduced exhaustion, and lowered tumor burden. Furthermore, IL-3 increases basophil-derived IL-4, elevating CTL IFN-γ production and viability. CTLs are vital as IL-3 has no impact on tumor growth in RAG1-/- mice or after CTL-depletion; moreover, IL-4-/- tumor mice do not benefit from IL-3 supplementation. This research unveils IL-3-mediated CTL-basophil crosstalk regulates anti-tumor immunity and offers the prospect of harnessing IL-3 as a promising approach for enhancing cancer immunotherapy.
The role of interleukin 27 in donor specific alloantibody responses
Authors: Valenzuela Jose , Fan Ran , Gorbacheva Victoria , Chuluyan Eduardo , Fairchild Robert , Valujskikh Anna 
Published: November 2024

Abstract
There is a great clinical need for new approaches inhibiting pathogenic donor specific alloantibodies (DSA) and antibody-mediated rejection (AMR) in transplantation. Targeting interleukin-6 (IL-6) showed modest promise for AMR treatment. Interleukin-27 (IL-27) also belongs to IL-6 cytokine family, utilizes the same receptor signaling chain, gp130, and has many overlapping functions with IL-6. The current study tested the role of IL-27 in humoral immune responses using mouse transplant models. First, B6.WT and B6.IL-27R-/- recipients (H-2b) were transplanted with BALB/c heart allografts (H-2d). By d. 21 posttransplant, B6.WT recipients developed serum DSA against donor class I and class II MHC. In contrast, DSA responses were markedly reduced in B6.IL-27R-/- recipients. We next tested how IL-27 neutralization affects DSA using mAbs against p28 IL-27 subunit. B6.WT recipients transplanted with BALB/c skin allografts and treated with mAb against either p28, IL-6, or IL-6R or control IgG for the first two weeks posttransplant. While control recipients developed anti-H-Dd DSA, none of anti-p28 mAb treated recipients generated DSA, comparable to the effects of anti-IL-6 or anti-IL-6R therapies. Anti-IL-27 mAb resulted in a more profound decrease in the numbers of antibody secreting spleen plasma cells compared to anti-IL6 and anti-IL-6R treatments. Our data identify IL-27 as a novel therapeutic target to inhibit pathogenic DSA responses in transplant recipients.
Sustained cross-talk between donor T cells and IL-33+ stromal cells maintains CD4 T cell differentiation and determines GVHD outcomes
Authors: Dwyer Gaelen , Mathews Lisa , Chalmers Bailey , Naaz Afsana , Poholek Amanda , Sacirbegovic Faruk , Shlomchik Warren , Turnquist Heth 
Published: November 2024

Abstract
Conditioning before allogeneic hematopoietic stem cell transplantation (AlloHSCT) increases the tissue injury signal IL-33 in fibroblastic reticular cells (FRC). Released IL-33 directly stimulates donor CD4 T cells to prime IL-12-independent Type 1 T helper cell (Th1) differentiation and expansion. Tissue stroma upregulates IL-33, but a role for IL-33 in sustaining the pathogenic donor Th1 responses causing GVHD is unclear. We compared B6 mice with inducible ST2 deletion (R26-CreERT2xSt2fl/fl) to wildtype (WT) R26-CreERT2 as T cell donors in a lethal GVHD model (B6 to BALB/c). Donor ST2 deletion at days 10-14 post AlloHSCT increased CD4 T cells Foxp3 expression with reciprocal decreases in Tbet expression in both the lymphoid organs and target tissues. Sustained IL-33 signaling also maintained donor T cell TCF1 expression. Ablating ST2 after GVHD development improved clinical scores and promoted recipient weight gain. How bioactive IL-33 is released from nuclear sequestration remains undefined. RNAseq analysis suggested that IL-33 stimulates T cell granzyme B (GzmB) expression and B6 GzmB deficient (Gzmb-/-) donor T cells displayed reduced activation and expansion similar to ST2 deficient CD4 T cells. In contrast to GzmBWT, anti-IL-33 antibodies had no impact on GzmBKO T cell responses. Thus, cross-talk between donor T cells and IL-33+ stroma orchestrates the T cell identities that are critical to sustain the pathogenic CD4 T cell responses causing GVHD.
Mechanism underlying why donor splenocyte transfusion helps costimulation blockade drive transplantation tolerance
Authors: Mora Ricardo , Xie Stephen , Stewart Emma , Cassano Alexandra , McIntosh Christine , Wang Peter , Wang Ying , Chen Luqiu , Chong Anita , Alegre Maria-Luisa 
Published: November 2024

Abstract
Solid organ transplantation is the preferred treatment for end-stage organ failure. Achieving stable donor-specific transplantation tolerance holds the potential to vastly improve the quality of life for transplant recipients by eliminating lifelong exposure to drug toxicity, a higher risk of malignancy and infection, and the development of chronic rejection associated with conventional immunosuppression. In mice, treatment with anti-CD154 (aCD154) prolongs graft survival, but the addition of donor splenocyte transfusion (DST) to aCD154 produces a robust state of graft-specific tolerance to fully MHC-mismatched cardiac allografts. In this study, we investigated parameters that dictate the efficacy of DST treatment. Our results support an antigen dispersal hypothesis whereby DST enables alloantigen to circulate to all secondary lymphoid organs where it can prime alloreactive T cells under cover of aCD154, and induce widespread tolerance in a larger repertoire of T cells than enabled by an allograft alone.
Modulating alloreactive immune responses by controlled dual delivery of Fas and IL-2R agonists using a lipocoacervate platform
Authors: Tedla Mebrahtu , Wright Nathaniel , Tarique Mohammad , Ulker Vahap , Yee-Flores Kathleen , Yolcu Esma , Wang Yadong , Shirwan Haval 
Published: November 2024

Abstract
Allogeneic islet transplantation is an effective treatment for type 1 diabetes (T1D). Graft rejection is controlled by immunosuppressive drugs that have adverse effects. An imbalance between Teffs and Tregs is the primary cause of T1D and graft rejection. Activated Teff cells express Fas and are sensitive to FasL-mediated apoptosis. Treg cells are relatively refractive to apoptosis and expand in response to IL-2. Thus, a combination of Fas and IL-2R agonists has the potential to modulate alloreactive responses for sustained graft survival. Given the off-target effects of systemic delivery of biologics, we established a lipocoacervate formulation consisting of a biodegradable polycation, PEAD, and polyanion heparin to form coacervate for controlled delivery of a novel Fas agonist, SA-FasL, and IL-2. Release kinetics and activities of proteins were assessed in vitro. The immunomodulatory efficacy of protein-loaded lipocoacervate was assessed in vivo. IL-2 and SA-FasL proteins showed a steady release over 30 days in vitro. IL-2 released on day 14 had minimal activity loss, whereas FasL showed ~20% activity loss on day 9. Treatment with SA-FasL/IL-2-loaded lipocoacervate modulated in vivo alloreactive T cell responses, resulting in an increased Treg/Teff cell ratio. In conclusion, lipocoacervate is an effective platform for controlled and sustained release of SA-FasL and IL-2 biologics to modulate allo and autoreactive T cells with significant therapeutic potential for T1D.
Understanding the role of neutrophils in Staphylococcus aureus-induced skin inflammation
Authors: Matsumoto Masanori , Nunez Gabriel 
Published: November 2024

Abstract
Staphylococcus aureus, a common pathogen in the skin, triggers skin diseases including atopic dermatitis and cutaneous abscesses. We recently found that neutrophils play differential roles in epicutaneous and intradermal S. aureus inoculation models. To understand the role of neutrophils in S. aureus-induced skin inflammation, neutrophil gene expression profiles were analyzed by single cell RNA-sequencing (scRNA-seq) analysis using those two infection models.
Methods: We infected C57BL6/J mice intradermally or epicutaneously with S. aureus USA300 strain and isolated neutrophils from the infected skin tissues followed by scRNA-seq analysis. To identify neutrophil genes that regulate skin inflammation, we generated specific gene deletion mice to investigate the underlying mechanisms.
Results: In the intradermal S. aureus infection model, we found that inducible nitrogen oxide synthetase (iNOS) gene expression was upregulated in neutrophils and that skin lesion size was significantly increased in neutrophil-specific iNOS-deficient mice. In the epicutaneous S. aureus inoculation model, however, the expressions of genes (padi4, mmp8/9, s100a8/9) required for neutrophil extracellular trap (NETs) were upregulated, and skin inflammation was suppressed in padi4-deficient mice.
Conclusion: These data suggest that neutrophil NO production is critical for dermal S. aureus killing, while NETs formation induces skin inflammation by epidermal S. aureus colonization.