bluuebunny/crossref_metadata_2025 huggingface dataset:
Taxation Policies as an Environmental Protection Instrument
Authors: Dias Sara Luís , Ribeiro João Sérgio
Published: August 2023

The “green taxation,” which usually includes taxes on energy, transport, pollution, and resources, is being used to achieve environmental and sustainability purposes. The European Union is promoting this strategy, creating different provisions related to the usage of taxes to change environmental behaviors. Portugal, as a member-estate of European Union, follows this path establishing a policy where taxes are being used for these purposes. However, “green taxation” is also a significant form of revenue and, currently, it is difficult to define the criteria for the creation of these taxes, as they are not considering the ability to pay (one of the main principles in the creation of taxes) affecting the equitable distribution of the tax burden. The authors can conclude that, with the masking of “taxes required for environmental protection,” these taxes are being used as an additional source of revenue, distorting the principles underlying the creation and collection of taxes.
The Portuguese Environmental Tax Burden
Authors: Oliveira Gisela Pinto , Carmo Cecília M. R. , Cruz Sérgio Ravara
Published: August 2023

Environmental taxation is an important tool in the hands of member states to combat against climate change and promote environmental sustainability. This chapter describes in detail the taxes that are part of the Portuguese tax system and that have the social function of protecting the environment. For this purpose, a background of the Portuguese Green Tax Reform is provided and each of the environmental taxes considered to be the main burdens of the Portuguese tax system are explained. This study aims to contribute to providing academics and practitioners with more information on environmental taxation and to motivate regulators to develop more articulated green taxation strategies in the future.
Adoption of Digital Art NFTs in Hong Kong
Authors: Wong Sze Wing , Chan Mimi Mei Wa , Chiu Dickson K. W.
Published: August 2023

The most popular uses of blockchain technology in the art market are related to non-fungible tokens (NFTs). This chapter explores the adoption of NFT in the digital art market and its future development. The authors explore NFT adoption in the digital art market with the five pillars of the digital entrepreneurship model, including knowledge base, business environment, finance, technology, and culture. Their scarcity and utilities determine the value of NFTs. Collectors and artists should also be aware of the benefits and drawbacks of blockchain technology and take appropriate steps to guarantee their rights are protected. This chapter provides a fundamental review of the current development and outlook of NFT and opens new opportunities for future study. Scant research focuses on the present condition and adoption of NFTs within the Hong Kong digital art market. This review offers a much-needed exploration and understanding, particularly beneficial for potential investors and participants seeking a comprehensive insight into NFT adoption.
Expression and purification of conserved, protective streptococcal antigens for monoclonal antibody isolation
Authors: Mahoney Lauren , Mousa Jarrod J
Published: August 2023

Streptococcus pyogenes is a significant cause of infectious morbidity and mortality globally. Antibiotics remain the essential treatment for S. pyogenes infection however, as resistance against long-used antibiotics grows amongst bacterial pathogens and antibiotic failure becomes increasingly common in S. pyogenes treatment, the necessity of alternative treatment methods escalates. Here, we have selected conserved streptococcal antigens to express in Escherichia coli in pursuit of isolating protective human monoclonal antibodies (mAbs) for streptococcal infection. We have successfully expressed the serine proteinase ScpC (180 kDa) which impedes the recruitment of neutrophils to the infection site. In addition, we have produced SpyAD (48.9 kDa), a protein that is conserved across streptococcal emm types and is shown to play an essential role in bacterial adhesion and division. Both proteins are important virulence factors and are highly conserved across emm types, making them promising targets for future vaccination and therapeutic treatments. Following the expression and purification of the antigens, SpyAD and ScpC are used to screen human peripheral blood mononuclear cells (PBMCs) for antigen specific B-cells via flow cytometry. This work confirms the use of E. coli as an expression system for streptococcal antigens and the employment of such antigens in protective mAb isolation.

Supported by the NIH Post-Baccalaureate Research Education Program
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

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

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

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

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

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

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

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

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

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

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

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

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.
Age specific differences in murine alveolar endothelial cells to pathological conditions
Authors: Liu Lunhua , Akkoyunlu Mustafa
Published: August 2023

Neonatal pulmonary viral infections can have diverse outcomes ranging from bronchiolitis and pneumonia in RSV infection to more subdued response in SARS-CoV-2 infection. In this study, we investigated the responses of murine alveolar endothelial cells (MLEC) to neonate mice serum in presence of TLR agonists-treated macrophages, which can mimic SARS-Cov2 induced innate cell activation. We found that when poly I:C and ssRNA40 activated macrophages were cocultured with neonatal mice serum-treated MLECs, the expression of TNFα and IL-6 were significantly decreased. Moreover, macrophage co-culture leads to decreased ACE2 and VEGFR2 in adult MLECs, but macrophage co-culture increased ACE2 expression in neonatal MLECs under Poly I:C but not ssRNA40 stimulation. Addition of neonatal mice serum to MLECs increases the reactive oxygen species but downregulates multiple inflammatory signal pathways. Concurrently, the expression VE-cadherin and Shp2 was downregulated in neonatal mice serum exposed MLECs, which leads to compromised MLEC barrier integrity. Morphologically, neonatal mice serum, but not adult mice serum, triggered the lipid droplets formation and upregulated SR-BI and CD36 receptor expression in MLECs. Taken together, neonatal and adult MLECs differentially regulate cytokine-secretion from activated macrophages, and neonatal serum breaks the tight cell junction between MLECs by down regulating Shp2 and VE-Cadherin expression and induces the lipid droplets formation in MLECs through receptors SR-BI and CD36. The differences in neonatal and adult MLEC responses to pathologic conditions may reflect the age specific pulmonary manifestations to viral infections.
The role of tumor necrosis factor receptor 2 (TNFR2) in EAE
Authors: De Oca Arena Maria Montes , Bartsch Brenden , Schwegman Lance , Abdul-Baki Nawal , Forsthuber Thomas
Published: August 2023

Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system which affects over 2 million people worldwide and is the most common neurological disability in young adults. MS is a heterogenous disease characterized by recurrent, remitting and/or progressive neurological deficits, and clinical manifestations include nerve damage, visual loss, motor and balance impairment, gait disturbance, and others. While several environmental and genetic risk factors have been identified, such as the DR2b (DRB1*1501) human MHC class II allele, the molecular mechanisms underlying the development and severity of MS remain poorly understood. Tumor necrosis factor is a cytokine that exerts its effects through two different surface receptors TNFR1 and TNFR2, and it has been identified as a key player in MS pathogenesis. TNFR2 is thought to have a protective role in experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, by altering the function of astrocytes and regulatory T cells (Tregs). Previous data from our lab demonstrated that in the absence of TNFR2, transgenic mice expressing the DR2b allele and lacking mouse MHC class II (DR2bDR2) develop a more severe and progressive form of EAE, exhibiting astrogliosis. We have previously demonstrated that multiple Treg-associated genes were upregulated in CD4+ T cells from DR2bDR2 mice compared to those from control mice. However, the exact mechanism by which TNFR2 regulates Treg function in EAE is unknown. Here, we show that deletion of TNFR2 impacts the ability of Tregs to expand in vitro as well as their localization within the brains of mice undergoing EAE. Our results provide key insights into the mechanism by which TNFR2 limits and protects against CNS inflammation.
Investigating a novel role of DRAK2 in T cell cytoskeletal regulation and synapse formation
Authors: Wilander Benjamin , Harris Tarsha L , Mandarano Alexandra H , Guy Cliff , Crawford Jeremy , McGargill Maureen A
Published: August 2023

DRAK2 is a serine/threonine kinase expressed highly in T cells and a potential therapeutic target for autoimmune disease. Drak2−/− mice are resistant to autoimmune disease in models of type 1 diabetes and multiple sclerosis. This resistance is partly attributed to diminished accumulation of autoreactive T cells in target organs of Drak2−/− mice. Interestingly, Drak2−/− T cells can effectively eliminate tumors and pathogens similar to wildtype T cells, which makes DRAK2 an ideal target to inhibit autoreactive, but not pathogen or tumor-specific T cells. Additionally, Drak2−/− T cells are hypersensitive to suboptimal stimulation, indicating that DRAK2 impacts T cell function. These changes in Drak2−/− T cell function may be due to alterations in T cell activation, survival, or migration. However, the distinct role of DRAK2 in T cells remains unclear. To address this, we conducted RNA-seq analysis on wildtype and Drak2−/− T cells at rest and following activation. We identified significant changes in expression of genes within pathways associated with synapse formation, and cytoskeletal regulation. Thus, we examined whether DRAK2 impacts the T cell cytoskeleton or synapse formation. Our data show that Drak2−/− T cells have significantly reduced polymerized actin, but not monomeric actin compared to wildtype T cells. Additionally, we observed that synapse area was significantly increased while the concentration of TCR in the synapse was decreased in the absence of Drak2. Drak2−/− T cells also had more malformed synapses and formed fewer conjugates with antigen presenting cells than wildtype T cells. Ongoing experiments will address how DRAK2 mechanistically regulates T cell actin polymerization and synapse formation.

Supported by ALSAC and St. Jude Graduate School of Biomedical Sciences.
Gads is differentially required for the thymic development of conventional and innate-like T cells
Authors: Yablonski Deborah , Shalah Rose , Hallumi Enas , Klopstock Naama
Published: August 2023

Thymocyte development is tightly regulated by TCR signaling. Gads bridges a TCR-inducible interaction between SLP-76 and LAT, two adaptors that are essential for T cell development. In contrast, Gads-deficient mice have moderately impaired thymic development and accumulate non-naive peripheral T cells of unclear origin.

To illuminate the regulatory roles of Gads, we developed a model for tamoxifen-induced ablation of Gads (GadsiKO), accompanied by the expression of tdTomato. Using this model, we followed the fate of GadsiKO (Tom+) and Gads+ (Tom−) thymocytes within the same mouse. Compared to Gads+ (Tom−) DN thymocytes, GadsiKO (Tom+) DN thymocytes were less likely to undergo conventional β-selection; yet their ability to develop into γδ or NKT cells was not impaired.

A key feature of thymocyte development is the TCR-induced upregulation of CD5, which occurs during β-selection and further increases upon positive selection. Nevertheless, most GadsiKO DP thymocytes did not express CD5, nor did they increase calcium upon TCR ligation. These observations suggest that a TCR-independent, innate-like thymic developmental pathway may be unmasked in the absence of Gads.

GadsiKO SP thymocytes were heterogeneous: some were CD5−TCRlo, while others were CD5+TCRhi. Among the CD5+ GadsiKO CD4 SP thymocytes, a large proportion exhibited innate-like characteristics such CD44hiCD62Llo and/or NK1.1+. In contrast, peripheral GadsiKO T cells maintained a naive CD44loCD69Lhi phenotype for up to five weeks. Our results suggest that Gads is dispensable for peripheral quiescence, but regulates thymic development, by promoting conventional αβ T cell development, while suppressing innate-like developmental fates.

Supported by a grant from the Israel Science Foundation, and by a short-term grant from the Rappaport Institute for Research in the Medical Sciences
Trisomy 8-associated Autoinflammatory Disease (TRIAD) is Characterized by Dysregulated Myeloid Cells
Authors: Manthiram Kalpana , Xu Qin , Wu Zhijie , Bowes Mary , Gao Shouguo , Oguz Cihan , Elkahloun Abdel , Kalsi Shelley , Dulau-Florea Alina , Romeo Tina , Shi Lihong , Cassini Thomas , Deuitch Natalie , Kirby Martha , Anderson Stacie , Bruns Deborah , Ombrello Amanda , Barron Karyl , Remmers Elaine , Donovan Frank , Peterson William , Cochran Jill , Laxer Ronald , Greenberg Jessica , Zein Wadih , Brooks Brian , Perazzio Sandro , Kone-Paut Isabelle , Al-Mutiari Nora , Thors Valtyr Stefánsson , Pálmadóttir Johanna Guðrún , Aeschlimann Florence , Quartier Pierre , Murphy Karen , Chandrasekarappa Settara , Torgerson Troy , Schwartzberg Pamela L , Kastner Daniel
Published: August 2023

Trisomy 8 mosaicism (T8M) has been associated with a Behçet’s-like inflammatory disease, but immunologic features and treatment responses are not well-characterized. Here, we characterize 20 individuals with constitutional T8M and inflammatory disease. Most participants had congenital dysmorphologies and developmental delay. Two developed hematologic malignancies, two had bleeding diathesis due to platelet dense granule deficiency, and most had macrocytosis. The majority had recurrent fever and severe oral ulcerations, while nearly half had genital ulcers or rash. Colchicine, apremilast, and IL-1 and TNFa inhibitors were effective therapies. With ddPCR on sorted cell populations, we found that cells from the myeloid lineage (monocytes, neutrophils, megakaryocytes, erythroid progenitors) had a significantly higher percentage of trisomy 8 mosaicism compared to those from the lymphoid lineage (T and B cells) in both the peripheral blood and bone marrow, suggesting that trisomy 8 is tolerated to a greater degree by myeloid cells. Furthermore, we found that participants with T8M had more classical monocytes and had upregulation of genes associated with activated neutrophils and monocytes in their whole blood compared to healthy controls. With single cell RNAseq, we identifed which cells were trisomy 8 and disomy based on chromosome 8 gene expression and found that trisomy 8 monocytes had distinct transcriptional signatures and alteration of innate immune genes. Our findings suggest that patients with T8M are prone to a distinct autoinflammatory disease which we propose calling trisomy 8 associated autoinflammatory disease (TRIAD) and have complications due to dysregulation of cells arising from the myeloid lineage.

This work was supported by the Intramural Research Program of NIAID, NHGRI, and NHLBI, NIH.
Deciphering the functional role of commensal-induced T-DC clustering along sensory nerves in the lung
Authors: Ichise Hiroshi , Hor Jyh Liang , Arroyo-Mejías Armando J , Belkaid Yasmine , Germain Ronald N
Published: August 2023

The airway mucosa is a primary site of the immune response to inhaled antigens. However, how immune homeostasis is maintained in this organ during continuous exposure to airborne particles and microbial antigens is not known. Previous studies have shown that the spatial organization of immune components in relationship to each other and to non-hematopoietic components in tissues is critical to their proper function, as is communication with commensal microbes. To interrogate such relationships in the lung, especially the possible connections between immune cells and the densely innervated pulmonary airway, 3D information is required. However, current methods for 3D imaging of optically cleared tissue do not provide the marker depth afforded by high-content 2D imaging.

To overcome this constraint, we combined a fast hydrophilic tissue clearing technique, Ce3D, with the recently developed IBEX technique, and created Ce3D-IBEX. To date, up to 20 markers have been visualized in a single sample. Using Ce3D-IBEX, we identified a previously uncharacterized clustering of T cells and dendritic cells in proximity to the pulmonary sensory nerves in naïve mice. Agonistic denervation of the sensory nerves with resiniferatoxin reduced the number of clusters, and the cluster formation was disrupted in germ-free mice as well as OT-I and OT-II TCR-Tg mice. These findings indicate that the clustering requires sensory nerve-derived signals and TCR-mediated recognition of bacterial antigens. Future studies will be aimed at elucidating the functional role of the clusters in homeostasis and disease conditions and determining the molecular basis for neuro-immune communication that regulates cluster formation and activity.

All studies were done with approval from the ICUC of the NIH/NIAID. This work was supported by the Intramural Research Program of NIAID, NIH.
Respiratory syncytial virus promotes allergic predisposition via gastrointestinal microbiome alteration
Authors: Ethridge Alexander Dale , Martinez Llilian Arzola , Rasky Andrew J , Huffnagle Gary B , Lukacs Nicholas W
Published: August 2023

Respiratory syncytial virus (RSV) is a leading cause of childhood hospitalizations and increases the relative risk for the development of wheezing and asthma following acute bronchiolitis. Previously, we observed increased allergic predisposition following early life RSV infection in response to cockroach allergen (CRA) with a concurrent alteration in the gastrointestinal microbiome in mouse models of RSV infection and allergy. Importantly, differences in the gastrointestinal microbiome have been reported in children at a high risk for asthma. However, the effect of viral infection induced gastrointestinal microbiome alteration on allergic predisposition has not been studied. Our initial studies showed that early life RSV infection led to altered microbiome development that corresponded with disease development. We hypothesized that RSV-induced alteration of the microbiome can predispose infected animals to enhanced allergic inflammation. To address this, neonatal germ-free mice were colonized with normal microbiome from specific pathogen free mice with subsequent RSV infection followed by CRA modeling at 4 weeks post-infection. Colonization of germ-free neonatal mice protected the development of severe allergic asthma later in life, while an early life RSV infection of colonized mice reduced the protective effect of the microbiome by increasing airway hyperreactivity and T helper 2 induced responses. Taken together, our data indicate that early life RSV infection alters the neonatal microbiome leading to enhanced allergic predisposition.

Supported by grants from NIH (R35 HL150682, R01 AI138348)
Ablation of regulatory T cells lead to compromised quality of humoral immunity to SA14-14-2 live attenuated Japanese encephalitis vaccine
Authors: Nikam Bhushan Sanjay , Kalia Anurag , Pandit Suvechchha , Gupta Nimesh
Published: August 2023

Japanese encephalitis (JE) virus is the leading cause of encephalitis in Asia and the western Pacific. Most people infected with JE do not develop symptoms or have only mild symptoms. However, few infected people develop encephalitis and about 1 in 4 cases are fatal. Currently there is no treatment for JE however the vaccines are available. One of the most widely used vaccine against JE is live attenuated SA14-14-2, which is recommended for children and adults. However, less is known about the mechanism by which the vaccine induces protective immunity. We previously found that vaccine-induced CD4+ T cells play crucial role both in protection and in the development of protective humoral immunity. Here, we attempted to understand the role of CD4+ T cell subsets, mainly regulatory T cells (Tregs), in the mechanism of protection conferred by the vaccine. To address this, we used the Treg depletion mouse model and the TCRb−/− mouse as challenge model. We observed that depletion of Tregs lead to enhancement in germinal centre (GC) reaction to vaccine. We found a significant increase in GC-Tfh cells and GC-B cells in Treg ablation condition. We also found an increase in the magnitude of antibodies in response to vaccine particularly the IgG1 isotype. However, as marked by FRNT and the challenge studies, the neutralizing potential of the antibodies was compromised in Treg depleted condition. Altogether, the data suggest that the exclusion of Tregs may enhance the cellular and humoral response, however, that will negatively impact the protective quality of humoral immunity. Overall understanding the immunological mechanism of protective immunity conferred by this vaccine will be essential key for rational design of vaccine against JE or other flaviviruses.

NII
Correlates of immunity in SARS-CoV-2 post-vaccine infections in the Prospective Assessment of SARS-CoV-2 Seroconversion (PASS) Study
Authors: Goguet Emilie , Weiss Carol , Olsen Cara , Powers John , Coggins Si’Ana , Tribble David , Davies Julian , Meyer William , Ansari Sara , Conner Tonia , Illinik Luca , Lusvarghi Sabrina , Edwards Margaret Sanchez , Jackson-Thompson Belinda , Hollis-Perry Monique , Pollett Simon , Wang Gregory , Alcorta Yolanda , Wong Mimi , Saunders David , Mohammed Roshila , Ortega Orlando , Parmelee Edward , Lindrose Alyssa , Haines-Hull Hannah , Moser Matthew , Samuels Emily , Tso Marana , Graydon Elizabeth , Malloy Allison M W , Schully Kevin , Burgess Timothy , Broder Christopher , Laing Eric , Mitre Edward
Published: August 2023

Background

We sought to determine pre-infection correlates of immunity against SARS-CoV-2 post-vaccine infections.

Methods

Serum and saliva samples from 176 BNT162b2-vaccinated adults in the Prospective Assessment of SARS-CoV-2 Seroconversion study were collected between October and December 2021 and assessed for serum and saliva levels of WT SARS-CoV-2 Spike (S)-specific IgG and IgA antibodies using a microsphere-based multiplex immunoassay. Serum samples were also assessed for neutralization activity against D614G, Delta (617.2), and Omicron BA.1 and BA.1.1 variants using a lentiviral pseudovirus neutralization assay. After the fall visit, participants reported all positive PCR and/or antigen tests for SARS-CoV-2. Duration, severity, and type of symptoms, as well as risk exposures and adherence to precautionary measures, were assessed by questionnaires during the spring 2022 visit.

Results

Thirty-two participants (18.2%) had infections between December 7, 2021 and April 1, 2022. Pre-infection anti-S IgG antibody levels and neutralizing titers against BA.1 and BA.1.1 were higher in individuals that did not develop infection (p values 0.0098, 0.0313, and 0.021, respectively). No individuals with an anti-S level greater than 15,000 binding antibody units (BAU/ml) developed a post-vaccine infection.

Conclusion

High serum anti-S IgG antibody levels, high neutralization titers against Omicron subvariants, and low home risk scores correlated with protection against post-vaccine infections during the initial Omicron wave. BAU levels greater than 15,000 in the fall of 2021 were associated with complete protection during the initial Omicron wave.

This work was supported in whole, or in part, with federal funds from the Defense Health Program (HU00012020067, HU00012020094) and the Immunization Healthcare Branch (HU00012120104) of the Defense Health Agency, United States Department of Defense, and the National Institute of Allergy and Infectious Disease (HU00011920111), under Inter-Agency Agreement Y1-AI-5072.