bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intranasal nanoparticle vaccine elicits protective immunity against Mycobacterium tuberculosis
Authors: Sefat K M Samiur Rahman , Kumar Monish , Kehl Stephanie , Leekha Ankita , Peniagua Melisa-Martinez , Spencer Charles M , Ouellet Hugues , Varadarajan Navin 
Published: August 2023

Abstract
Mucosal vaccines have the potential to elicit protective immune responses at the point of entry of respiratory pathogens thus preventing even the initial seed infection. Unlike licensed injectable vaccines, mucosal vaccines comprising protein subunits are only in the development stage. One of the primary challenges associated with mucosal vaccines has been the identification and characterization of safe yet effective mucosal adjuvants that can effectively prime multi-factorial mucosal immunity. In this study, we tested NanoSTING, a liposomal formulation of the endogenous activator of the stimulator of interferon genes (STING) pathway, cyclic guanosine adenosine monophosphate (cGAMP), as a mucosal adjuvant. Intranasal administration of this vaccine provides protection comparable to subcutaneous administration of the live attenuated Mycobacterium bovis vaccine strain Bacille–Calmette–Guérin (BCG) upon challenge with aerosolized Mycobacterium tuberculosis Erdman strain. Protection against the bacterium is likely due to CXCR3+ lung resident T cells that are known to be important for the control of bacterial infection. Intranasal delivery also induces IFNγ secreting CD4+ T cells in lung which is necessary for intracellular bactericidal activity. Our results indicate that NanoSTING adjuvanted protein vaccines can elicit a multi-factorial immune response that provides durable protection from infection by M. tuberculosis.
The 1000 mouse experiment: quantifying the variability of tumor response to CAR T cell therapy
Authors: Kenet Adam , Achar Sooraj , Pouzolles Marie , Su Angela , Kondo Taisuke , Dwivedi Alka , Qin Haiying , Chien Chris , Mirazee Justin , Ishii Kazusa , Jia Dongya , Taylor Naomi , Altan-Bonnet Grégoire 
Published: August 2023

Abstract
Chimeric Antigen Receptor (CAR) T cell therapy is a type of cancer immunotherapy designed to target specific antigens on the surface of tumor cells using engineered T cells. While effective, there is often variability in patient responses and even within an experiment given the same experimental conditions. Analyzing this variability may help identify key modalities to successful CAR T therapy. We present a platform to explore this variability by quantifying the luminescence signal from in vivo tumor growth over time in a mouse model of human CAR T response. We developed a Python toolkit (https://github.com/soorajachar/radianceQuantifier) to automate labeling, cropping, quantification, and plotting of bioluminescent tumor image data. Applying this toolkit to digitize dozens of prior in vivo experiments conducted over several years allowed us to generate a dataset consisting of over 1000 mice. We then performed statistical analyses and mathematically modeled the time dynamics of tumor responses to CAR T cells in this dataset.
We identified three distinct phases of tumor response: initial tumor growth, tumor decay due to killing by CAR T cells, and tumor relapse. These phases were fit to differential equations to extract the growth and decay rates of the tumor for each mouse. Upon fitting our model to the experimental data, we found that the growth rate of relapsed tumors was slower than the initial tumor growth rate within individual mice, suggesting a selection of edited tumor cells post CAR T cell therapy. Future work will leverage our quantitative framework to explore the alterations between relapsed tumor cells and those that are eradicated by CAR T cells.
Supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute (Bethesda, MD)
Gene co-expression regulatory analysis associated with tertiary lymphoid structures and endometrial cancer progression
Authors: Clark Alexis , Lillard James W 
Published: August 2023

Abstract
Endometrial cancer (EC) is the most common cancer diagnosis of the reproductive organs among women in the United States. Previous studies have shown the chemokine CXCL13 was correlated with forming B-cell and other immune cell aggregates in the presence of high endothelial venules (HEV). These ectopic lymphoid formations are referred to as tertiary lymphoid structures (TLS). Some studies have shown that TLS are associated with a reduced risk of recurrence and improved immune response in cancers, while others have described negative cancer outcomes associated with TLS. Few studies have focused on the role of TLS in EC and the pathways involved in tumorigenesis. In this project, we identify chemokine-mediated gene networks associated with TLS formation and EC pathogenicity. We obtained RNA-Seq EC samples (n=643), which included Type I Uterine Corpus Endometrial Cancer (UCEC, n=587) and Type II Uterine Carcinosarcoma (UCS, n= 56) from the Cancer Genome Atlas (TCGA). Hierarchically clustered genes were correlated, functionally annotated for mRNA, and evaluated using a weighted gene network co-expression analysis (WGCNA). Functional enrichment analysis, such as Gene Ontology (GO) and the database for annotation, visualization, and integrated discovery (DAVID), were used to identify and visualize biological functions associated with the hub genes overrepresented in significant modules. Modules associated with metastasis and survival are representative of novel and known pathways. The analysis reveals that CXCL13 may be involved in cell cycle progression and apoptosis pathways. This study provides novel insights into the biological pathways associated with TLS, EC tumorigenesis, and patient survival risks.
Supported by Frederick National Laboratory for Cancer Research subcontract IDIQ NCI (75N91019D00024) and MSM/TU/UABCCC Partnership NCI (U54CA118638)
Multiplexed RNA-protein co-detection to explore tumor-immune landscape using a novel automated RNAscope&[trade] assay
Authors: Dikshit Anushka , Basak Sayantani , Kim Ge-Ah , Yu Julia , Deshpande Sonali , Wang Li-Chong , Srinivasan Maithreyan 
Published: August 2023

Abstract
Development of next-generation immunotherapies need a highly sensitive multiomics platform to evaluate diagnostic and therapeutic biomarkers. Tissue heterogeneity poses immense challenges to understanding underlying molecular mechanisms using techniques such as qRT-PCR or bulk sequencing. Single-cell RNA sequencing can provide information about precise cellular composition of tissues, data analysis can be cumbersome and spatial context is lost. With single-cell spatial platforms such as RNAscope, target gene and protein expression can be visualized to characterize cell types and tissue neighborhoods. Here, we demonstrate a novel method for the simultaneous detection of RNA and protein using a modified co-detection assay.
This novel co-detection assay enables visualization of a combination of up to 12 RNA and/or protein targets on the same sample. We used a set of antibodies targeting key immune and tumor cell markers-PD1, CD3, CD4, CD8, CD68, FOXP3 and KRT17, along with RNA biomarkers to interrogate the tumor microenvironment (TME) in human FFPE tumor samples.
Using a combination of RNA and protein targets, we characterized different cell types such as T cells, macrophages, and tumor cells in the TME. We also developed a novel method for visualizing intercellular interactions between PD1 and PD-L1, offering insights into popular checkpoint mechanisms that are targeted for therapeutic intervention in cancer treatment.
The assay offers a powerful technique for visualizing target RNA biomarkers in specific cell-types identified by cell-marker protein expression. This is a valuable tool for multiomic analysis and accurate interrogation of complex tissues to obtain insights into novel biomarkers and therapeutic targets.
Duocarmycin SA reduces proliferation and induces apoptosis in Acute Myeloid Leukemia cells in vitro
Authors: Chen William , So Leena , Jawhar Yasmeen , Williams Terry G , Drew Natalie , Nguyen Nhi , Casiano Carlos A , Dovat Sinisa , Boyle Kristopher E , Francis-Boyle Olivia L 
Published: August 2023

Abstract
Acute myeloid leukemia (AML) is a blood cancer that is characterized by the clonal expansion of immature myeloid blasts. Duocarmycin SA (DSA) is an antitumor antibiotic that induces DNA alkylation and has demonstrated cytotoxic activity against several cancers, but not AML. Preliminary data from our lab demonstrated that DSA induced cytotoxic effects on AML cells in vitro. The objective of this study was to investigate the mechanism by which DSA exerts its cytotoxic effects on AML cells in vitro. We hypothesized that DSA will induce cell cycle arrest and contribute to cellular apoptosis. The human AML cell line (Molm-14) was used for all studies and the phenotype of the cells was confirmed by flow cytometry. To test our hypothesis, we performed dose response (MTT), proliferation (CFU and EdU) and apoptosis (Hoffman Modulation and Annexin V/7-AAD) assays. Our results showed that the AML cells were CD45+, CD33+, CD13+, CD14+ and CD4+ and the IC50 of DSA was ~11 pM. DSA induced cell cycle arrest at the G2/M phase, significantly decreased the production of colonies and significantly reduced the proliferation of AML cells in a dose-dependent manner (63.5%, 37.6%, 14.3% and 2.9% of EdU+ cells following treatment with 0, 20, 100 and 500 pM of DSA respectively for 96 hours). Finally, DSA also increased apoptosis of AML cells in a dose-dependent manner (4.5%, 9.4%, 56.7%, and 92.0% at 0, 20, 100 and 500 pM DSA respectively following treatment with DSA for 72 hours). In summary, picomolar concentrations of DSA induces cell cycle arrest, reduces proliferation and colony formation, and induces apoptosis as a mechanism of cell death in AML cells. Thus, highlighting DSA as a potential therapeutic candidate for further evaluation on AML cells.
This work was supported by funding from the School of Pharmacy at Loma Linda University.
Using spectral flow cytometry and BD Horizon RealYellow™ and BD Horizon RealBlue™ Reagents to dissect the expression of metabolic proteins in resting and activated T cells
Authors: Baracho Gisele Vanessa , Gao Guo-Jian , Widmann Stephanie , Tyznik Aaron J 
Published: August 2023

Abstract
In response to activation, T cells undergo metabolic adaptations to meet the energetic and biosynthetic demands required for their rapid cell growth and proliferation. T cell receptor (TCR) and CD28 costimulation trigger the initial metabolic changes that support T cell activation including increase in glucose uptake. PI3K signaling is critical for upregulation of Glut1, the main glucose receptor in T cells. In this study, we describe an assay for detection of Glut1 and key metabolic enzymes using fluorochrome-conjugated antibodies that have been validated for flow cytometry. Our analyses showed a broad expression of Glut1 and other metabolic markers across human immune cell populations. In T cells, Glut1 was expressed in resting T cells and upregulated in response to anti-CD3/CD28 stimulation. Glut-1high cells were found among CD69+ activated T cells, which also showed higher expression levels of glucose-6-phosphate dehydrogenase (G6PD) compared to the CD69− cells. Notably, the PI3K inhibitor Ly294002 abrogated the upregulation of Glut1 and G6PD expression in activated CD69+ T cells. Ly294002 cell treatment did not affect the expression of hexokinase 1 (HK1), CD69, PD-1 or CD25, suggesting that PI3K inhibition impacted the expression of select metabolic markers and not overall T cell activation. These results support literature evidence showing the importance of the glycolytic pathway on T cell activation while demonstrating optimized experimental conditions for resolution of multiple metabolic markers. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
BD, BD Horizon RealBlue and BD Horizon Real Yellow are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved.
H3K9 dimethyltransferase G9a is an important epigenetic modulator of B cell differentiation
Authors: George-Alexander Lou-Ella , Kania Anna K , Scharer Christopher D , Boss Jeremy M 
Published: August 2023

Abstract
Plasma cell differentiation is a tightly regulated process coordinated by the timed expression of several transcription factors as well as histone modifying enzymes that modulate chromatin accessibility. During this process, the histone methyltransferase (HMT) G9a, dimethylates histone H3 lysine 9 (H3K9) at promoters and inhibits gene expression through the recruitment of proteins that impair chromatin accessibility. HMTs are expressed ubiquitously but display distinct enzymatic activities and patterns of chromosomal localization. During plasma cell differentiation, G9a was found to co-localize with Blimp-1, which is required to silence genes associated with a B cell fate and cellular proliferation. However, the processes that are modulated by G9a mediated dimethylation during plasma cell formation remain to be elucidated. To study the role of G9a in plasma cell differentiation, we crossed G9afl/fl mice onto the CD19Cre/+ background (G9aKO mice). Stimulation of CD19Cre/+ (CreCtrl) and G9aKO mice with the T cell independent antigen LPS resulted in a significant increase of activated B cells and plasmablast in G9aKO mice. Further characterization of this phenotype, identified a skewing of the mature B cell sub-populations in G9aKO mice, accompanied by altered proliferation rates when challenged. ATAC-Seq and RNA-Seq will be used to identify chromatin accessibility and expression changes in G9a deficient mice. The CUT&Tag assay will also be used to validate regions that are subject to direct modulation by G9a during plasma cell differentiation. Together, our data suggests that G9a contributes to regulating proliferation during plasma cell differentiation.
his work is supported by grants from NIH/NIAID (RO1 AI123733 and P01 AI125180 to JMB)
B cell Tet2 promotes maturation of the antibody response through active DNA demethylation and OGT-mediated histone glycosylation of Aicda and Prdm1 loci
Authors: Li Shili , Peng Yibo , Li Xin , Yang Zhimin , Zan Hong , Casali Paolo 
Published: August 2023

Abstract
Epigenetic DNA methylation regulates gene expression and, therefore, cellular functions. It typically represses gene transcription, which can be re-activated by demethylation in the absence of cell division by the recently characterized Tet1, Tet2 and Tet3 dioxygenases. Tet2, the most abundant Tet protein in B cells, not only actively demethylates DNA but also promotes Ogt recruitment to chromatin to effect Ogt-dependent histone O-GlcNAcylation. This potentiates catalytic DNA demethylation to activate gene expression. To define the role of Tet2 in B cell functions and antibody responses, we generated AicdacreTet2fl/fl mice, which specifically delete Tet2 in activated B cells. AicdacreTet2fl/fl mice showed significantly reduced antibody responses to T-dependent and T-independent antigens, due to downregulation of AID and Blimp-1 expression, CSR/SHM and plasma cell differentiation. Accordingly, activated AicdacreTet2fl/fl B cells reduced DNA hydroxymethylation (resulting in increased methylation) and histone O-GlcNAcylation of Aicda and Prdm1 promoters and enhancers, thereby dampening CSR/SHM and plasma cell differentiation. These were partially rescued by enforced expression of Tet2 mutants defective in catalytic activity or Ogt-binding, but not by a double mutant lacking both activities. Accordingly, Tet2 and Ogt inhibitors synergistically inhibited Aicda and Prdm1 expression in both human and mouse B cells. Conversely, Tet2 activation by vitamin C enhanced CSR and plasma cell differentiation. Thus, Tet2 plays a B cell-intrinsic role in modulation of CSR/SHM and plasma cell differentiation through catalytic active DNA demethylation and (non-catalytic) Ogt-mediated histone O-GlcNAcylation.
Supported by NIH grants AI 079705, AI 105813/105813S1, AI138944, AI 167416 and Lupus Research Alliance grant LRA 641363 to PC.
Dynamics of lymphoid-mammary epithelial cell interaction during pregnancy and lactation
Authors: Yong Daniel , Corral Dan , Belkaid Yasmine 
Published: August 2023

Abstract
Breast milk has been shown to play a key role in the transfer of immunity from mother to offspring. Antibodies and lymphoid cells can be passed through breast milk and impact offspring immunity. However, the role of these lymphoid cells in lactation and in the control of offspring immunity remains unclear. We observed that the lymphoid cells present in the breast milk are distinct from those found in the blood. Compared to the blood, breast milk T cells are enriched in a T-bet+ unconventional population. Imaging analysis revealed that these T-bet+ cells gradually accumulate in the mammary gland during pregnancy and are specifically localized around and inside the mammary epithelium. We will utilize confocal and intravital imaging of the mammary gland in virgin, pregnant, and lactating mice to characterize the dynamics of this mammary-associated T-bet+ population throughout pregnancy and lactation. Understanding the dynamics of T-bet+ cells and their anatomical localization within the mammary gland will help us decode how these cells impact the remodeling of the mammary epithelium during pregnancy and lactation. We propose that the remodeling of the mammary gland imparted by the immune system can also influence the transfer of immunity from mother to offspring, thereby affecting the development of offspring immunity.
This work was supported in part by intramural funds of NIAID, NIH.
Analysis of intestinal epithelial cell responses to IFN-γ during Cryptosporidium infection
Authors: Pardy Ryan D , Walzer Katelyn , Striepen Boris , Hunter Christopher A 
Published: August 2023

Abstract
Cryptosporidium species are intracellular parasites that infect intestinal epithelial cells (IEC) and the rapid cycle of growth, cell lysis and reinfection every 12 hours can cause severe enteric disease in young or immunocompromised patients. Interferon-γ (IFN-γ) has a crucial role in protective immunity and mice with an IEC-specific deletion of the IFN-γ receptor (IFN-γRΔIEC) are highly susceptible to infection. However, the dynamics of IFN-γ signalling to IEC and how this leads to parasite control remain poorly understood. Based on the use of a reporter mouse for IFN signalling in vivo, while macrophages showed a transient response to treatment with IFN-γ, IEC exhibited a sustained response to IFN-γ that peaks at 24h. Indeed, the treatment of mice with recombinant IFN-γ transiently limits parasite shedding but takes 24h to show protective activity. Single-cell RNA sequencing of enterocytes from naïve and infected mice identified upregulation of several IFN-γ-inducible effectors but highlighted that induction of these genes was comparable in infected and bystander IEC. Together, these results suggest that while Cryptosporidium-infected cells are responsive to IFN-γ signalling, the protective effects of IFN-γ may be mediated through activation of uninfected bystander enterocytes. These studies provide new insights into the dynamics and function of IFN-γ signalling on IEC that are broadly relevant to other IEC-restricted pathogens.
Supported by grants from NIH (R01 AI148249) and post-doctoral fellowships from the Canadian Institutes for Health Research and Fonds de Recherche du Québec – Santé.
Characterization of Mouse Anti-Crude Peanut Extract IgE Monoclonal Antibodies
Authors: Waritani Takaki , Cutler Dawn , Agosto Ibis , Lomax Sidney M , Chang Jessica 
Published: August 2023

Abstract

INTRODUCTION
Immediate hypersensitivity reactions to peanuts, an IgE-mediated food allergy, are a major public health concern. For allergic patients, avoidance currently remains the only viable option. Mouse peanut allergy models are used for studying pathogenesis and therapeutics, However, establishing animal models with high IgE antibody titers is difficult.

OBJECTIVE
Newly developed mouse anti-crude peanut extract (CPE) IgE monoclonal antibodies (mAbs) were evaluated with in-vitro and in-vivo assays used for studying the pathogenesis of and therapeutics against peanut allergies.

METHODS and RESULTS
Four hybridomas that produce IgE mAbs against CPE were established from BALB/c mice orally administrated CPE with cholera toxin. All mAbs recognized the Ara h1 allergen in CPE by western blot and worked dose dependently in sandwich ELISAs using an anti-IgE capture mAb and biotinylated CPE. However, only three mAbs worked in indirect ELISAs with CPE coated plates, depending on individual epitope specificities. The three mAbs degranulated RBL-2H3 cells and their degrees of activation varied by mAbs and concentration. In female BALB/c mice, two mAbs developed footpad delayed type hypersensitivity, which peaked at 1–2 hours after sensitization and drop in body temperature in anaphylaxis reactions, which peaked at 40–60 minutes after an IV injection of CPE.

CONCLUSION
Two out of four IgE mAbs can work for both in-vitro and in-vivo peanut allergy studies. Particularly, the mAbs can develop allergic reactions with sensitization alone in mice. This will be especially useful to evaluate therapeutic methods associated with mast cell activation as the mAbs can accelerate studies with no lengthy immunization protocols.
B1-like B cells derived from the bone marrow and not fetal-derived B1 B cells are responsible for the protective IgE induced in helminth infection
Authors: Zellner Matthew , Urban Joesph F , Conrad Daniel , Martin Rebecca 
Published: August 2023

Abstract
Nippostrongylus brasiliensis (Nb) is the strongest natural inducer of IgE. Despite this fact, asthma rates have an inverse correlation in countries endemic with parasitic infection, and multiple groups have shown that helminth infection has a protective phenotype in murine asthma. In a previous publication our lab showed that innate-like B1 B cells produced IgE in response to Nb infection and this IgE inhibits helminth clearance in a mast cell (MC) dependent manner. Therefore we hypothesized that the reduction in helminth clearance and the protective phenotype in asthma was driven by B1 B cell IgE preventing MC degranulation. To test this, we utilized a four week model of murine asthma and injected IgE anti-DNP as our “benign” IgE bi-weekly. Treatment significantly improved the outcomes associated with this model. Excited by these data we sought to further define the transcriptional differences between B1 and B2 B cells before and after Nb infection using RNAseq analysis. We found that gene expression in peritoneal (PL) B2 B cells were more similar to PL B1 B cells than to lymph node resident B2 B cells, both before and after Nb infection. Next, we sought a better way to separate B1 and B2 B cells than traditional cell surface markers. Utilizing a lineage specific reconstitution model where fetal derived B1 B cells made FLAG-tagged IgE and bone marrow (BM) derived B2 B cells made untagged IgE, we demonstrated that fetal derived B1 B cells were not responsible for Nb-induced IgE. Using the same reconstitution system, we showed that BM derived B cells that express B1 B cell surface markers were a significant source of Nb-induced IgE. Overall, we show that protective Nb-induced IgE is produced by B1-like B cells derived from the BM and not fetal-derived B1 B cells.
Funding: This work was supported by funding from VCU’s CTSA (UL1TR002649 from the National Center for Advancing Translational Sciences) to R.K.M. and the CCTR Endowment Fund of VCU as well as funding from NIAID-NIH (R56AI139658) to RKM. Services used were supported, in part by the NIHNCI Cancer Cancer Support Grant P30 CA016059.