bluuebunny/crossref_metadata_2025 huggingface dataset:
Premene krutosti v sodobni ideološki krajini (kot jo redefinira vojna v Ukrajini)
Authors: Klepec Peter
Published: August 2023

Tekst se loteva problematike krutosti izhajajoč iz tega, kako ta nastopa v vsakdanji vladajoči ideologiji. Če hočemo izmeriti pulz naše sodobne ideološke krajine, potem ne moremo mimo tega, da jo je nedavno močno pretresla ruska agresija na Ukrajino. Vojna v Ukrajini je bila večkrat označena za kruto, nasploh je krutost nekaj, kar danes soglasno velja za nekaj zavrženega in odvratnega. A za takšno velja tudi mučenje, ki se ga načelno preganja, skrivoma pa izvaja. Enako velja za vojno, ki soglasno velja za kruto. Kaj torej pomeni izjava »vojna v Ukrajini je kruta«, v kakšne kontekste se vpisuje? Tekst se tega vprašanja loteva na več ravneh, ki segajo od sodobnega pojmovanja vojne, krutosti, širšega zgodovinskega konteksta, v katerem izjava nastopa, pa do vprašanja ali to pomeni, da smo v nekem pomenu z eno nogo še vedno trdno v dvajsetem stoletju. Navezujoč se na Badioujevo delo Stoletje, ki izpostavlja vlogo strasti do realnega, vojne (ki naj bi končala vse vojne) in krutosti v dvajsetem stoletju, ta izjava pomeni tudi, da dvajsetega stoletja v nekem pomenu (še) ni konec in da bo morda z vojno v Ukrajini (znova) to stoletje (do)končno obračunalo s samim seboj.
Examining the Link Between Corruption and Bank Credit
Authors: Yakubu Ibrahim Nandom , Bunyaminu Alhassan , Musah Alhassan
Published: August 2023

This research examines the influence of corruption on the expansion of bank credit in Sub-Saharan Africa from 2000 to 2017. By utilizing the generalized method of moments (GMM) technique, the investigation reveals a significant positive correlation between corruption and bank credit. Additionally, the findings indicate that while stability in the banking sector leads to a decrease in bank credit, economic growth and inflation contribute significantly to the growth of bank credit. Based on these results, the study offers essential recommendations to foster integrity and responsible lending practices in the banking sector. It proposes that banks establish robust whistleblowing procedures, encouraging employees to promptly report any instances of corrupt transactions associated with credit advancement. Through the cultivation of a transparent and accountable culture, banks can effectively address and mitigate the adverse impacts of corruption on credit growth. Furthermore, the research advocates for the enforcement of strict non-price constraints to regulate lending practices.
Carbon Tax
Authors: Dias Paulo Jorge , Rocha Andreia
Published: August 2023

One of the main climatic concerns is the increase of CO2 concentrations in the atmosphere. In the attempt and need to combat this problem, several regulatory policies have been applied in several countries by the governments, with the carbon tax being one of the most economical and effective policies in reducing CO2 emissions, thus leading to an increase in scientific research on this topic. Therefore, the main objective of this study is characterizing the existing research on the theme Carbon Tax. A sample of 121 scientific articles was collected, where several characteristics related to the content of published articles and respective authors were subsequently identified. The results obtained reveal that the number of publications has been increasing, and the themes most related to this investigation are: cap-and-trade, carbon dioxide emissions, climate change, computable general equilibrium (cge) model and emissions. Regarding the profile of researchers, the type of collective authorship is the most common and the country with the most affiliated authors is China.
The Role of Sustainability Reporting in Corporate Tax Transparency
Authors: Monteiro Sónia , Guzmán Beatriz Aibar , Sánchez Isabel-María García
Published: August 2023

Tax transparency is often associated with standardized reporting. Among the goals of the 2030 agenda, goal 12.6 encourages organizations to adopt sustainable practices and integrate sustainability information into their reporting cycle. The Global Reporting Initiative (GRI) published the GRI 207- Tax (2019), aiming to increase transparency and accountability in corporate tax practices, by providing disclosure guidance for companies to disclose information about their tax strategy, governance, and risk management. As it is a recent standard, research on GRI-207 is still scarce. Thus, this chapter aims to contribute to the debate on the link between corporate reporting and fiscal responsibility by providing an overview on the background and structure of the GRI 207, which could serve as a basis for future research in the field of tax disclosure, as well as contributing to the policy debate. This chapter could have political and practical implications, as transparency in the disclosure of tax-related issues in corporate reporting could mitigate the reputational tax risk of companies.
Application of AI in Academic Library Services
Authors: Donkor Antonia Bernadette , Afrane Daniel Akwasi
Published: August 2023

Academic libraries are mandated to support the information needs of their students, staff, and faculty. In the current technological environment and the influx of artificial intelligence technology into services provision and delivery, this study assessed the knowledge and perception of librarians on the use of AI in library services provision in Ghana. The population for the study consisted of librarians from all 15 public universities in Ghana. Findings from the study revealed that the librarians were knowledgeable of the emergence of AI and sources their information on AI mainly from research articles. It was also revealed that educational level had a significant positive (r= 0.3, p<0.01) relationship with the sources of knowledge on AI tools and applications. There was a positive significant relationship (r= 0.533, p<0.01) between educational level and the frequency of knowledge acquisition on the application of AI in academic library services provision. Academic libraries are encouraged to invest in AI tools and applications to leverage their advantages.
A novel self-adjuvanted mRNA vaccine expressing the Antigen-85B-p25 epitope of Mycobacterium tuberculosis protects mice against tuberculosis
Authors: Mishra Abhishek , Singh Vipul , Court Karem Pinto , Godin Biana , Sukhovershin Roman , Kiss Daniel , Khan Arshad , Jagannath Chinnaswamy
Published: August 2023

Mycobacterium tuberculosis (Mtb) is a leading cause of death due to infections. BCG is the most used vaccine despite having partial efficacy against the tuberculosis. We have developed a synthetic mRNA vaccine, SelmRp25-C5, which expresses the p25 epitope of Mtb-derived Antigen-85B and a TLR-2 stimulating C5 adjuvant peptide from CFP-10 protein. We evaluated its efficacy both ex vivo and in mice.

Methods

Mouse and human macrophages (MΦ) and mouse dendritic cells (DCs) were treated with SelmRp25-C5 vaccine or EGFP-mRNA control followed by antigen presentation assays and assessment of cytokine secretion. C57Bl/6 mice were vaccinated subcutaneously with two doses (5μg per mouse) of SelmRp25-C5 vaccine, or EGFP-mRNA and one dose of subcutaneous BCG (106 CFU per mouse) as a control, followed by aerosol infection with Mtb. Four weeks after Mtb challenge, mice were sacrificed, and organs were analyzed for Mtb CFU, RNAseq, Ag85B-p25 tetramer-specific CD4 T cells and memory T cells.

Results

SelmRp25-C5 vaccine induced robust antigen presentation to T cells in both mouse MΦs and DCs and in human MΦs, and enhanced Th1 cytokine secretion. Two doses of SelmRp25-C5 vaccine protected mice by reducing Mtb burden by 1log10. Protection induced by SelmRp25-C5 vaccine was associated with an increase in Ag85B-p25-specific CD4 T cells, increased IFN-γ and IL-2 secreting T cells and CD4 effector memory T cells. Lung and spleens of protected mice showed an upregulation of Spleen tyrosine kinase (Syk) and genes of the C-type lectin receptor-pathway compared to control mice.

Conclusions

We propose that the new generation self-adjuvanted mRNA vaccine strengthen both innate and adaptive immunity to provide protection against tuberculosis.

Supported by NIAID, NIH AI122070, AI138587; AI161015.
Structure-guided vaccine design to focus antibody responses to neutralizing epitopes on dengue virus envelope protein
Authors: Thiono Devina J , Samaras Demetrios , Phan Thanh T N , Tian Shaomin , Forsberg Lawrence J , Kuhlman Brian , de Silva Aravinda
Published: August 2023

The four dengue virus (DV) serotypes are mosquito-borne flaviviruses responsible for dengue fever. Live-attenuated DV vaccines that are in human trials have had mixed results with efficacy for just one or two serotypes and an increased dengue risk in some vaccinated populations. DV recombinant envelope (E) proteins as subunit vaccines have had limited success, likely due to DV wild type E protein (WT rE) is mostly monomeric at physiological temperature. Hence, it loses the quaternary structure epitopes recognized by neutralizing human antibodies (Ab) on the native dimer. We previously showed that with targeted amino acid mutations, we can form DV thermostable dimers (SD rE) displaying native quaternary epitopes targeted by strongly neutralizing Ab. Here we report on studies to test if Ab response can be directed to these quaternary epitopes by using SD rE as vaccine antigens. Since fusion loop (FL) Ab can cause antibody dependent enhancement (ADE) that increases disease severity upon heterologous DV infection, we also tested SD rE with mutated FL. Mice were immunized with DV2 WT rE or SD rE variants to compare Ab properties induced by each antigen. We found that SD-vaccinated mice had higher DV2 functional Ab titer compared to WT-vaccinated. To map vaccine responses, we run neutralization assays with chimeric epitope transplant DV and sera depleted of Ab subpopulations. We use ADE assay to determine if stabilized E dimer and mutated FL reduces ADE compared to WT rE. Preliminary results indicate that WT-vaccinated mice neutralizing Ab bind to simple epitopes on rE monomers, while SD-vaccinated mice had mostly dimer-specific neutralizing Ab. Our results demonstrate the promise of structure-guided design for developing dengue subunit vaccines.

NIH/NIAID U19 AI109784

R01AI107731

DOD/US Army Medical Research ACQ W81XWH1820034
Pre-existing T cell immune response specific to computationally optimized broadly reactive hemagglutinin antigens (COBRA-HA) in influenza vaccinees
Authors: Moraes Vanessa S , Souquette Aisha , Ross Ted M
Published: August 2023

The biggest challenge in developing a universal influenza vaccine is the extensive antigenic drift. In contrast to humoral immunity, cell-mediated immunity is more cross-reactive and can protect against various subtypes and drift variants. Our research group has described computationally optimized broadly reactive antigens (COBRA) to develop HA-based broadly protective influenza vaccine. However, studies that characterize the potential of COBRA-HA to elicit a functional T-cell response have yet to be developed. Cross-reactive response to conserved T cell epitopes from previous exposure can provide an efficient heterologous immunity. Thus, we aim to immune profile the pre-existing and cross-reactive COBRA-HA T cell response at the functionality and epitope conservation level to identify potential targets to be included in universal flu vaccine design. To achieve this goal, 50 donors who received the seasonal influenza vaccine (Fluzone, Sanofi) for two consecutive seasons were HLA typed, and the epitopes of COBRA-HA vaccine candidates Y2 (H1) and NG2 (H3) were predicted using IEDB software. Using the high dimensional flow cytometry, eight peptides for Y2 and 15 for NG2 were combined in pools to determine the phenotypes and cytokine secretion. We found that the vaccination induced a cross-reactive COBRA HA-specific CD4+ and CD8+ T cell response independent of antibody titers. Next, we will evaluate the Th helper subsets (Th1/Th2/Th17/cTfh) and the breadth of immune response by deconvoluting the pools in IFNγ Fluospot assay. Nonetheless, the next generation of the universal influenza vaccine recalled a functional CD4+ pre-existing response, demonstrating the potential efficacy of COBRA-HA in clinical trials.

National Institute of Allergy and Infectious Diseases (NIAID), U.S. National Institutes of Health (NIH), Department of Health and Human Services contract 75N93019C00052 (TMR).
Characterization of immune response towards modified Coxsackie B virus-like particle vaccine in a murine model
Authors: Soppela Saana Marika , González-Rodríguez Martin , Stone Virginia , Kummola Laura , Lampinen Vili , Jouppila Niila , Laitinen Olli , Flodström-Tullberg Malin , Junttila Ilkka , Hankaniemi Minna
Published: August 2023

Coxsackievirus Bs (CVBs) are common human RNA viruses that belong to the enterovirus genus of Picornaviridae. Most commonly, infections with these viruses manifest with mild flu-like symptoms. However, CVBs are also known to cause severe diseases, including aseptic meningitis, myocarditis, and chronic dilated cardiomyopathy (DCM), encephalitis, pancreatitis, and hand-foot-and-mouth disease. In this study, we aimed for a versatile characterization of a modified CVB virus-like particle vaccine in a murine model. The modified CVB-VLP was produced using baculovirus-insect cell production platform. Purified and characterized vaccine was administered to mice via s.c. or i.m. routes, either with or without an adjuvant. Immunological characterization of vaccine response included both humoral and cellular characterization. Antigen-specific IgG levels and neutralizing antibodies in the sera were measured to determine the humoral immune response. To characterize the cellular immunity, activation markers of the lymphocytes were identified using flow cytometry. Additionally, the cytokine secretion of the stimulated splenocytes was determined with FluoroSpot assay. The modified CVB-VLP induced high levels of IgG and neutralizing antibodies in the sera of vaccinated mice. Further, the in vitro stimulation of splenocytes induced activation of mainly CD4+ T-cells, and adjuvanted vaccine induced a significant production level of IFN-γ and IL-2. These results indicate that the virus-like particle combined with an adjuvant is a promising vaccine candidate. In addition, our current challenge study with coxsackie B1 virus will shed us more insight on the protective role of this vaccine.

Supported by grants from Finnish Foundations (Instrumentarium Science Foundation, Paulo Foundation and Ida Montini Foundation)
Immunogenicity of intravenous irradiated or auxotrophic BCG immunization in macaques
Authors: Robertson Molly A , Lehman Chelsea C , Buscan Allison N , Dahlvang James D , Darrah Patricia A , Roederer Mario , Seder Robert A
Published: August 2023

Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), is a leading cause of death worldwide, particularly in people with HIV. The current vaccine against TB, BCG, is a live-attenuated strain of M. bovis that is administered intradermally at birth in endemic countries. BCG protects children against disseminated TB but fails to protect adolescents and adults against pulmonary TB, the most transmissible form. Thus, more effective TB vaccines are urgently needed. We previously demonstrated that delivering BCG intravenously (IV) elicits a high number of Mtb-specific T cells in the airway, the site of infection, and confers robust protection against Mtb challenge 6 months later in rhesus macaques. While whole organism vaccines provide broad antigenic breadth, safety concerns may limit clinical development of a replication-competent IV BCG regimen, especially in immunocompromised individuals. To address this, we vaccinated macaques IV with either irradiation-inactivated (Ird) BCG or a live, replication-incompetent auxotroph (Aux) of BCG and compared their immunogenicity in the bronchoalveolar lavage (BAL) to wild type (WT) BCG. Early results indicate that Aux BCG elicits a similar magnitude of Mtb-specific T cell responses in the BAL compared to WT BCG; in contrast, Ird BCG is less potent in generating such responses. These studies provide data on whether whole organism vaccines need to be live, persist, or replicate to confer durable immunity against TB.

Funded by the NIH Intramural Program
Investigating tissue-resident memory T cell differentiation of CAR T cells reveals an unexpected role for CD69
Authors: Cheng Menglin , Bergsbaken Tessa
Published: August 2023

Adoptive cell therapy using chimeric antigen receptor-expressing (CAR) CD8+ T cells has revolutionized the treatment of liquid tumors; however, CAR T cells have had more limited success against solid tumors. CD8+ tissue-resident memory T cells (Trm), which are characterized by surface expression of CD69 and CD103 and a unique transcription factor circuit, are found within solid tumors and associated with improved tumor control. However, it is unclear whether CAR T cells adopt Trm features in the tumor microenvironment and how enhancing Trm differentiation of CAR T cells might impact their efficacy against solid tumors. Using the murine tumor model MC38, we identified both CD69+CD103- and CD69+CD103+ subsets that express Trm signature genes. To examine if CAR T cells follow a similar differentiation pattern, human carcinoembryonic antigen (hCEA)-specific CAR T cells with either a CD28 or 4-1BB costimulatory domain were transferred into hosts bearing MC38-hCEA tumors. We found the CD28 domain drove expansion of the CAR T population while delaying Trm marker expression compared to 4-1BB and non-CAR T cells. The persistence of a CD69-CD103-subset of CD28 CAR T cells strongly correlated with the number of hCEA-CD28 CAR T cells within the tumor. CD69-CD103- hCEA-CD28 CAR T cells were more proliferative and expressed lower levels of surface markers associated with dysfunction, including PD-1 and LAG3. We hypothesized CD69 could negatively regulate T cell proliferation and function; indeed, deletion of CD69 in hCEA-CD28 CAR T cells enhanced their proliferation and accumulation within tumors. In summary, hCEA-CD28 CAR signaling delayed Trm differentiation and CD69 expression in solid tumors resulting in prolonged T cell proliferation.

NIH 7R01AI153096
TCR/Chimeric Antigen Receptor (CAR) Cross-Antagonism to Fine-Tune CAR-T cell Immunotherapy
Authors: Altan-Bonnet Grégoire , Kondo Taisuke , Bourassa François X , Achar Sooraj , DuSold Justyn , Cespedes Pablo , Wahlsten Madison , Gaud Guillaume , Love Paul E , Dustin Mike , François Paul , Taylor Naomi
Published: August 2023

Chimeric antigen receptor (CAR) T cells, are created by extracting T cells from a cancer patient, engineering them to express a CAR targeting a tumor specific molecule, then reintroducing them back into the patient. A patient’s T cells contain their own endogenous T cell receptors (TCRs) however, which could potentially interact with the exogenous CAR inserted into the cell. In this study, we examine how TCR and CAR signals interact upon CAR-T activation. We show that weak TCR stimulation can reduce (antagonize) or increase overall CAR-T response, both in vitro and in vivo, across multiple tumor models, in both mouse and human T cells. We further show that the behavior of these TCR/CAR interactions can be manipulated by changing various characteristics of the TCR, CAR, and associated ligands. While this behavior is complex, we show that it can be described by a single mathematical model based on the adaptive kinetic proofreading scheme of ligand discrimination. We conclude by presenting potential applications for cancer immunotherapy.

Intramural Research Program of the National Cancer Institute
IL-33 promotes cardiovascular lesions development in a mouse model of Kawasaki Disease
Authors: de Carvalho Thacyana Teixeira , Jena Prasant Kumar , Franklin Michael Kyle , Fishbein Michael C , Crother Timothy R , Chen Shuang , Shimada Kenichi , Arditi Moshe , Rivas Magali Noval
Published: August 2023

Kawasaki Disease (KD), a pediatric acute febrile illness and systemic vasculitis, is the leading cause of acquired heart diseases in children. Coronary artery aneurysms (CAAs) occur in up to 25% of untreated children, which is reduced to 5% with intravenous immunoglobulin (IVIG) treatment. However, up to 20% of KD patients are refractory to IVIG and at higher risk of CAAs, highlighting the need to characterize the mechanisms mediating KD vasculitis to develop more efficient therapies. IL-33 is released upon inflammation and tissue damage and exerts its effects by binding to its receptor ST2 (IL1RL1). Levels of IL-33 are elevated in KD patients during the acute phase. However, how IL-33 contributes to the development of KD cardiovascular lesions remains unknown. Using the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis, we observed increased Il33 mRNA expression in hearts and abdominal aortas aneurysms of LCWE-injected mice. While ST2 was detected by immunofluorescence in the abdominal aortas of both control and LCWE-injected mice, IL-33 was mainly detected in tissues from LCWE-injected mice. Single-cell RNA-sequencing of abdominal aortas from LCWE-injected mice indicated that Il33 transcripts were expressed primarily by fibroblasts, endothelial cells, B cells, and vascular smooth muscle cells. In contrast, Il1rl1 was highly expressed by tissue infiltrating regulatory T cells, macrophages, eosinophils, and neutrophils. Blocking IL-33 with either anti-IL-33 antibody or using Il33−/− mice significantly attenuated LCWE-induced KD vasculitis. Our results indicate that IL-33 promotes LCWE-induced KD vasculitis in mice and could therapeutically be targeted to inhibit KD cardiovascular lesions.
IL-17 Receptor A protects against Helicobacter pylori- induced gastric cancer
Authors: Algood Holly Marie Scott , Brackman Lee C , Dixon Beverly R E A , Bernard Margaret , Jung Matthew S , Joshi Nikhita , Green Emily H , Piazuelo M Blanca
Published: August 2023

Infection with Helicobacter pylori is the predominant risk factor for the development of gastric cancer. Risk is enhanced by specific H. pylori virulence factors, diet, and the inflammatory response. Chronic activation of T helper (Th) 1 and Th17 pathways contribute to inflammation. Previous work suggests that while IL-17 activates several antimicrobial components of the innate immune response, IL-17 is also required to prevent a hyper-inflammatory adaptive immune response. To investigate if IL-17 receptor signaling contributes to the development of carcinogenesis, the transgenic InsGAS mouse, which is susceptible to H. pylori-induced gastric cancer, was used. InsGAS mice and InsGAS Il17ra−/− mice were infected with a cag T4SS positive strain for up to 6 months. By 6 weeks post infection, IL-17RA deficiency led to increased bacterial burden and increased gastritis, concomitant with an exacerbated Th1/Th17 cytokine profile, increased B cell infiltration and enhanced production of IgA despite reduced Pigr expression. Paragastric lymph nodes of InsGAS Il17ra−/− mice, while enlarged relative to InsGAS mice, exhibited altered gene expression profiles and potential loss of fibroblastic reticular cells. Gastric pathologies, including intramucosal carcinoma and adenocarcinoma with submucosal invasion, developed more frequently by 3 and 6 months post infection in H. pylori-infected InsGASIl17ra−/− mice. These increases in severe gastric disease were accompanied by loss of parietal cells, loss of mucin-producing cells and enhanced expression of ceacam1. These data suggest that IL-17RA signaling activates a protective pathway to prevent excessive inflammation which can drive gastric carcinogenesis after H. pylori infection.

This work was supported in part by Merit Review I01BX000915 (Algood) from the U.S. Department of Veterans Affairs, Biomedical Laboratory Research and Development Service. We acknowledge the Translational Pathology Shared Resource supported by NCI.NIH Cancer Center Support Grant 5P30 CA68485-19. Core Services performed through Vanderbilt University Medical Center’s Digestive Disease Research Center supported by NIH grant P30DK058404.
Immunomodulators identified via high-throughput screening enhance control of vaccine adjuvanticity
Authors: Rosenberger Matthew Glenn , Kim Jeremiah , Esser-Kahn Aaron
Published: August 2023

Stimulation of the innate immune system is crucial in both vaccinations and immunotherapies. This is often achieved through adjuvants, molecules that usually activate pattern recognition receptors (PRRs) and stimulate two innate immune signaling pathways: the nuclear factor kappa-light-chain-enhancer of activated B-cells pathway (NF-κB) and the interferon regulatory factors pathway (IRF). Engineering the immune response via fine control of these pathways, however, is quite difficult. We demonstrate the ability to alter and improve adjuvant activity via the addition of small molecule “immunomodulators” to existing PRR agonists. By modulating signaling activity instead of receptor binding, these molecules allow the customization of select innate responses. We demonstrate both inhibition and enhancement of the products of the NF-κB and IRF pathways by several orders of magnitude. Some modulators apply generally across many receptors, while others focus specifically on individual receptors. Modulators boosted correlates of a protective immune responses in an inactivated flu vaccine and reduced correlates of reactogenicity in a subunit typhoid vaccine. These modulators have a range of applications: from adjuvanticity in prophylactics to enhancement of immunotherapy. This approach to adjuvanticity offers greater control over early immune activation and provides researchers with a new tool to tailor end immune responses.

NIH 75N93019C00041
Immunological consequences of CD91 Single Nucleotide Polymorphisms in patients with sarcomas
Authors: Sedlacek Abigail L , Binder Robert J
Published: August 2023

Tumor immunosurveillance is critically dependent on several immune cell types and proteins including CD91. CD91 is expressed on the surface of antigen presenting cells (APCs) and binds a select few heat shock proteins (HSPs) released by dying tumor cells. These HSPs carry tumor antigens and initiate anti-tumor immune responses in de novo tumors. We have previously shown among multiple sarcoma types, osteosarcomas are poorly infiltrated by T cells and poorly immune edited. This correlates with reduced expression of CD91 on the surface of intratumoral APCs. CD91 is a highly polymorphic gene within the human population. Over 3500 single nucleotide polymorphisms (SNPs) have been identified, despite the relatively high conservation of CD91 throughout evolution. We have identified 155 of these SNPs within our sarcoma patient population, 11 of which are exonic and non-synonymous. We analyzed the immunologic data we obtained from patients with non-synonymous SNPs to patients with normal CD91 protein sequences. In addition, we used the computational modeling programs SIFT and PolyPhen-2 to predict the effects of these mutations on CD91 protein folding. SNPs rs746886209 and rs758644665 were indicated by both SIFT and PolyPhen-2 to have deleterious effects on CD91 folding and patients with these mutations had below average immune infiltration. While these data are currently correlative, we have developed an in vitro model to obtain proof of concept.
Using split-pool combinatorial barcoding to simultaneously characterize the transcriptome and TCR diversity of 1 million T cells
Authors: Papalexi Efthymia , Kim Grace , Hariadi Bryan , Schroeder Sarah , Matulich Peter , Tran Vuong , Diaz Daniel , Roco Charles , Rosenberg Alexander
Published: August 2023

Recent advances in single-cell sequencing allow for simultaneous profiling of T cell receptors (TCRs) and transcriptomes leading to the characterization of key T cell subsets with pathogen recognition and clearance capabilities. Despite their success, these methods rely on microfluidic devices or plate-based protocols with limited sensitivity and throughput (1,000s–10,000s of cells) making the study of disease-relevant T cells time consuming and costly.

To overcome these limitations, we have extended our split pool combinatorial barcoding technology to simultaneously characterize the TCRs alongside the full transcriptomes of up to 1 million T cells. As a proof of concept experiment, we applied our method to 920,000 primary T cells from 8 healthy human donors to characterize their TCR diversity. Applying our method, we recovered TCR sequences from at least one chain in 88% of T cells (807,000 cells). Furthermore, we detected hundreds of thousands of unique alpha and beta chains across all donors and found that most of these chains represent rare clonotypes detected in 1 or 2 cells. Lastly, we performed transcriptome-based clustering analyses to identify all major T cell subsets and discovered donor and subset-specific hyper-expanded clonotypes that could help us reconstruct each donor’s recent history of infection.

In summary, we report on the extension of our highly flexible and scalable combinatorial barcoding technology to allow researchers to profile up to 1 million T cells in a single experiment and investigate their functional responses during infection, cancer, autoimmunity, or therapeutic interventions.
Conserved epigenetic hallmarks of T-cell age
Authors: Zebley Caitlin C , Mi Tian , Soerens Andrew , Alli Shanta , Vasandan Anoop , Hines Christopher , Mueller April , Baylin Stephen , Jones Peter , Goldstein Harris , Mullighan Charles , Masopust David , Youngblood Ben A
Published: August 2023

As the immune system ages, the protective capacity from established immunological memory wanes over time and is often referred to as immune senescence. Yet the molecular hallmarks of lymphocyte age and replicative senescence remain largely undefined. Here we report our use of a multi-lifetime murine model of repeat vaccination to define a core epigenetic signature coupled to memory T cells that evade replicative senescence. Through adoptive transfer and boosting over a period of approximately four mouse lifetimes, memory T cells became enriched for repressive DNA methylation programs at cell cycle regulators including the canonical senescence gene P19Arf (CDKN2A/2B locus – human P14ARF). The senescence-resistance program was progressively acquired throughout phycological and non-physiological aging of murine T cells, leading to the development of a lymphocyte epigenetic clock that can be used to accurately measure a T cell’s age. Application of this clock in physiologically aged human polyclonal and CMV-specific T cells correlated with age-estimates using established epigenetic chronologic clocks. Moreover, T-cell clock-associated senescence-resistance programs were highly enriched in human malignancies of lymphoid origin, specifically delineating known T-cell acute lymphoblastic leukemia (T-ALL) subtypes and revealing non-physiological aging of HOXA9 and MLL pediatric leukemias (estimated to be ~100 years old). Discovery of the senescence-resistance epigenetic clock provides a novel molecular hallmark for describing physiological and non-physiological lymphocyte age.
Tissue-specific heterogeneity of Human T follicular helper cells marked by unique transcriptomes and regulatory architecture
Authors: Powell Michael Duane , Rose James R , Guopta Mansi , Rahmberg Andrew , Scharer Christopher D , Boss Jeremy M
Published: August 2023

T follicular helper (TFH) cells represent a distinct lineage of CD4 T cells that engage with cognate B cells, facilitating the production of high affinity vaccine- and pathogen-specific antibodies. In humans, TFH cells can be detected in several tissues throughout the body, each representing a unique cellular environment complete with distinct signaling molecules. Despite TFH cell importance to human health, it remains unclear if and how anatomical location results in diversity among TFH cells. Here, we detail tissue-specific heterogeneity of human TFH cells reflected in both transcriptome and chromatin architecture. TFH cells sourced from circulating peripheral blood (cTFH), lung lymph nodes (lnTFH), tonsils (tTFH), and an in vitro differentiation model (exTFH), were subjected to RNA-sequencing and ATAC-sequencing. When comparing expression of conical TFH genes, including BCL6 and PDCD1, a spectrum in magnitude emerged: cTFH < exTFH < lnTFH < tTFH. Increased chromatin accessibility at proximal cis-regulatory elements occurred within the loci of TFH genes in a similar tissue-dependent fashion and correlated with transcript levels. Additionally, unique gene signatures were detected in each TFH population, including the presence of distinct transcription factor networks, implying tissue specific TFH functionality. Integrating PageRank and scRNA-seq of tTFH cells revealed BCL-6, ASCL2, TOX, TOX2, and AIOLOS as potential regulators of the TFH gene program. Collectively, these findings detail tissue specific diversity amongst human TFH cells, while highlighting key developmental networks which could influence the design of future immunological therapies targeting TFH cells.

Supported by grants from NIH: (RO1 AI113021, F32 AI161857)
Interferon-beta mediated STAT4 up-regulation are distinct in the ex vivo human CD4+ T-cells priming dendritic cells
Authors: Tangtanatakul Pattarin , Makarathut Pannaphan , Phonboon Terdkiat , Amornsupak Kamolporn , Makchareon Jiradej , Ritparchak Patcharee , Hirankarn Nattiya , Sodsai Pimpayao
Published: August 2023

STAT4 (Signal Transducer Activator of Transcription factor 4) polymorphisms are risk factors contributed to autoimmune disease. While STAT4 has a dominant role in promoting interferon-gamma (IFN-g) production in T and NK cells, its existence and function exert in human dendritic cells (DCs) are still unknown. To characterize the STAT4 expression in human peripheral DCs, we isolated DCs from buffy coat of health blood donors (n = 4). The ex vivo DCs were enriched and sorted into 3 subtypes including plasmacytoid DCs (pDCs, CD123+), CD8+ T-cells priming DC (cDC1, CLEC9A+), and CD4+ T-cells priming DC (cDC2/3, CD1c+) through negative selection bead and flow cytometry. The presence of STAT4 as well as its potential target genes (interleukin-12, IL-12 and interleukin-23, IL-23) expression were examined in IFN-beta induced DCs maturation compared to immature DCs, using Real-Time PCR. We found the proportion of DCs subsets in peripheral mononuclear cells which are 0.05%, 0.02% and 0.3% for pDC, cDC1 and cDC2/3, respectively. The IFN-beta enhanced DC maturation markers (HLA-DR+ CD86+), especially in cDC2/3. Interestingly, up-regulated STAT4 expression (2.5-fold) were significantly prominent in mature cDC2/3 compared to immature (p < 0.01), while IFN-beta showed no influence on STAT4 expression in the cDC1 and pDC (p > 0.05). Interestingly, elevated IL-12 and IL-23 expression were significantly observed in activated cDC2/3 (p < 0.05). Further study to investigate the role of STAT4 in human cDC2/3 are needed. Our result firstly discover the existing of STAT4 expression in the ex vivo peripheral human DC.

Supported by Chulalongkorn University (Fundamental Fund, CU_FRB65_hea (84)_179_37_09).
The role of interleukin-12-PRMT5 axis in generating suppressive Th1-like induced regulatory T cells
Authors: Jadon Nidhi
Published: August 2023

After activation, naïve CD4 T cells can differentiate into T helper cell subsets (Th1, Th2, Th17, and Follicular helper T cells) or into regulatory T cells (Tregs), depending on the type of antigen-presenting cells (APC) and cytokine milieu encountered during differentiation. Recent studies suggest that CD4+Foxp3+ Tregs can produce the pro-inflammatory cytokine IFNg when stimulated in a Th1 cytokine environment, such as in the presence of IL-12, and stimulation under these conditions makes iTregs sensors for inflammatory cytokines.

The stability and suppressive functions of the iTregs depend on multiple factors. The goal of this study is to explore an epigenetic enzyme protein arginine methyltransferase 5 (PRMT5) and to understand the significance of this protein in the development of Th1-like iTregs. PRMT5 can either represses or activate transcription of target genes via symmetric di-methylation of arginine residues on histones. However, the role of PRMT5 in the differentiation of Th1-like iTregs has not been thoroughly investigated.

We demonstrate that differentiating iTregs in a Th1 cytokine environment generated a specific population of Th1-like iTregs with increased suppressive function. In Th1-like iTregs, we observed symmetric arginine di-methylation on histone (H3R2me2s) mediated by PRMT5, while this epigenetic marker is absent in iTregs. We also demonstrate that si-RNA mediated knockdown of PRMT5 reduced the suppressive capacity of Th1-like iTregs. Therefore, we hypothesize that the IL12-PRMT5 axis is crucial for developing suppressive Th1-like iTregs. Goals of further research are to generate stable immunosuppressive iTregs for therapeutic use.
Rab4A controls the overexpression of CD38 and depletion of IL-2 in CD4+ T cells: Potential involvement in proinflammatory lineage development in systemic lupus erythematosus
Authors: Park Joy Seonghee , Patel Akshay , Krakko Daniel , Nolan Jessica , Perl Andras
Published: August 2023

Rab4A is a GTPase that is overexpressed in systemic lupus erythematosus (SLE) patients’ T cells and enhances cell surface receptor recycling. Increased expression of CD38 and loss of IL-2 production have independently been associated with proinflammatory T cell development in SLE. We investigated the impact of Rab4A on the expression of CD38 and the production of IL-2.

Our lab created Rab4A-mutant Jurkat cell lines, containing GFP-expressing vector alone (control), doxycycline-inducible vectors that overexpress Rab4A (Rab4Ahi) or the dominant-negative mutant Rab4AS27N (Rab4ADN). They were co-stimulated with anti-CD3 antibody and PMA. After 24 hours, cell surface markers and cytokines were analyzed by flow cytometry and protein levels by western blot. NAD+ levels were measured by LC-MS/MS.

In the Rab4Ahi cells compared to the control, CD38 expression was 2-fold upregulated (p=2.49×10−13), intracellular production and secretion of IL-2 were significantly decreased (fold change=−0.566, p=1.16×10−7 and fold change= −0.481, p=0.0401, respectively), NAD+ concentration was decreased (fold change=0.647, p=0.0039), while pSTAT3 levels were 2-fold increased (p=0.0318).

CD38 is an NAD+ hydrolase, which regulates Sirtuin-1 activity, a NAD+-dependent histone deacetylase that suppresses STAT3 activity. Elevated pSTAT3 levels may underlie diminished IL-2 production by binding to the promoter of FoxO1, which inhibits IL-2 production. The overexpression of Rab4A and CD38, increased pSTAT3, and diminished IL-2 production reflect changes observed in SLE patients. Our results suggest that increased expression of Rab4A may underlie the overexpression of CD38 and diminished secretion of IL-2 in SLE.
Human OTULIN mutations can cause distinct inflammatory disease phenotypes depending on the protein domain that is mutated
Authors: Gil Deb , Liu Qi , Dang Elizabeth , Bhattad Sagar , Markle Janet
Published: August 2023

Linear ubiquitination is a post-translational modification that controls many immune signaling pathways. Linear ubiquitin chains are added to targets by LUBAC and removed by OTULIN, thus the opposing actions of LUBAC and OTULIN are critical for immune homeostasis.

To understand mechanisms of how ubiquitination controls inflammation, we leveraged the use of biospecimens from patients with a novel bi-allelic OTULIN mutation. These patients have pyoderma gangrenosum (PG), an extremely rare inflammatory skin disease. OTULIN mutations have been previously described to cause a systemic autoinflammatory disease called ORAS, yet these patients define a new monogenic disease. We sought to understand this discrepancy through functional analyses of OTULIN mutations that culminate in PG vs ORAS.

ORAS-causing OTULIN mutations impair the catalytic domain. However, the mutation in PG patients affects the domain responsible for binding LUBAC. Further, OTULIN’s ability to downregulate linear ubiquitin or NF-kB activity was unaffected. Patients’ transcriptional signatures showed elevated expression of genes involved in neutrophil activation and antigen presentation, and reduced expression of genes involved in Th17 differentiation, TLR signaling, and NOD-like receptor signaling. Deep immunophenotyping by CyTOF revealed that PG patients have higher frequencies of T cells and subtle defects of B cells and myeloid cells. These patients’ unique phenotype suggests an unrecognized function for OTULIN in skin inflammation. This discovery adds to the emerging spectrum of human immune-mediated diseases caused by defects in the ubiquitin pathway.
Impacts of the A20 protein polymorphism, rs2230926, in human B cells
Authors: Sutton Rachel Elizabeth , Gupta Mansi , Scharer Christopher D
Published: August 2023

Patients with autoimmune diseases, such as systemic lupus erythematosus (SLE), often have skewed B cell population ratios, accumulation of autoantibodies, and an increased amount of proinflammatory cytokines aiding in disease development. Genome-wide association studies (GWAS) have strongly linked polymorphisms in A20, encoded by TNFAIP3, to autoimmune diseases including SLE. A20 functions to inhibit the activation of nuclear factor kappa B (NF-κB) through ubiquitination and deubiquitylation of key signaling proteins and has been extensively studied in epithelial cells in response to TNF. Given the role of autoantibodies in SLE and the importance of NF-κB in regulating B cell fate, the role of SLE linked polymorphisms in A20 and their effects on both the canonical and noncanonical NF-κB pathways in human B cells is poorly understood. Our lab has identified an A20 polymorphism, rs2230926 (F127C), that is enriched in a cohort of African American females with SLE. Herein, we investigate the impact of rs2230926 on B cell function. We employed CRISPR/Cas9 genome-editing and generated a TNFAIP3 knockout lymphoblastoid Raji human B cell line. To determine how the polymorphism rs2230926 affects the NF-kB pathway, we re-introduced WT A20, A20-rs2230926, and a truncated version of A20 into the TNFAIP3-KO cell line. Using a combination of qRT-PCR, western blotting, and flow cytometry, we determined how rs2230926 influences the expression of proinflammatory cytokines, response to various B cell stimuli, and ultimately NF-κB activity. Dissecting A20’s role in NF-kB signaling will help to understand the dysfunction and regulation of B cells in autoimmunity.

Supported by grants from NIH to CDS (R01 AI148471)
Commensal Bacteria Maintain a Qa-1b-restricted Unconventional CD8+ T Population in Gut Epithelium
Authors: Guan Jian , Valerio Michael Manoharan , Peske J David , Park Chansu , Robey Ellen A , Sadegh-Nasseri Scheherazade , Shastri Nilabh
Published: August 2023

Intestinal intraepithelial lymphocytes (IELs) are characterized by an unusual phenotype and developmental pathway, yet their specific ligands and functions remain largely unknown. Here by analysis of QFL T cells, a population of CD8+ T cells critical for monitoring the MHC I antigen processing pathway, we established that unconventional Qa-1b-restricted CD8+ T cells are abundant in intestinal epithelium. We found that QFL T cells showed a Qa-1b-dependent antigen-experienced phenotype in the spleen and small intestine of naïve wild-type mice. The splenic QFL T cells showed innate-like functionality exemplified by rapid response to cytokines or antigen, while the gut population was refractory to stimuli. Microbiota was required for the maintenance, but not the initial gut homing of QFL T cells. Interestingly, monocolonization with Pediococcus pentosaceus, which expresses a peptide that cross-activated QFL T cells, was sufficient to maintain QFL T cells in the intestine. Thus, microbiota is critical for shaping the Qa-1b-restricted IEL landscape.

RO1AI130210, R37AI060040
Protective colonic iNKT cells are expanded by Goblet cell-associated antigen passages in CD1d-dependent manner in adults
Authors: John Vini , Barrios Bibiana E , Udayan Sreeram , Floyd Alexandria N , Joyce Elisabeth S , Schill Ellen M , Knoop Kathryn A , Blumberg Richard S , McDonald Keely G , Newberry Rodney D
Published: August 2023

Invariant natural killer T (iNKT) cells are unique innate immune cell which secrete cytokines and immune mediators. These cells recognize self or microbial glycolipids presented in the context of CD1d. iNKT cells can contribute to host protection or pathogenesis during intestinal inflammation. Colonic iNKT cells are established under the influence of the microbiota in early life and the current understanding is that colonic iNKT cells are fixed and not manipulable in later life. Goblet cells (GCs) or/and goblet cell associated passages (GAPs) deliver luminal antigens and maintain peripheral T regulatory cells in the steady state. However, GAPs in the colon are absent in adult mice due to GCs microbial sensing. We found that the glycolipids can be delivered through GAPs and that inducing GAPs in the colon in adult mice resulted in significant iNKT cell expansion. Deletion of GCs or deletion of CD1d on GCs prevented iNKT cell expansion suggesting a role for colonic GCs in presenting glycolipids to iNKT cells. Single cell RNA sequencing of colonic-iNKT cells showed significantly increased iNKT2 and iNKT1 subsets after colonic GAP induction. Proteomic analysis of these iNKT cells further confirmed the characteristics of expanded iNKT subsets. Moreover, iNKT cells expanding after colonic GAP induction persisted and were protective in DSS-induced colitis. Adoptive transfer of the expanded colonic iNKT cells protected in the DSS-induced colitis model, indicating that expanded colonic iNKT cells were sufficient for protection in this model. These findings indicate that colonic iNKT cells are not fixed during early life but can be expanded in a GAP dependent and GC CD1d dependent manner and can be protective in some models of colitis.

Crohn’s and Colitis Foundation
Low IL-13Ra1 Expression on Mast Cells Tunes Them Unresponsive to IL-13
Authors: Salomaa Tanja , González-Rodríguez Martín Ignacio , Kummola Laura , Hiihtola Lotta , Junttila Ilkka
Published: August 2023

Cytokine-mediated mast cell regulation enables precise optimization of their own pro-inflammatory cytokine production. During allergic inflammation, Interleukin(IL)-4 regulates mast cell functions, tissue-homing and proliferation, but the direct role of closely related IL-13 for mast cell activation remains unclear. Previous work has shown that mast cells are potent IL-13 producers, but here we show that mouse mast cells do not directly respond to IL-13 by Stat6 activation, as they do not express measurable amount of IL-13Ra1 mRNA. Consequently, IL-4 responses are mediated via type I IL-4R (IL-4/IL4Ra/gC) and IL-4-induced Stat6 activation is abolished in gC deficient mast cells. Type II IL-4R deficiency (IL-13Ra1 KO) has no effect on IL-4-induced Stat6 activation. In basophils, both IL-4 and IL-13 induce Stat6 activation in WT and gC deficient cells, while in type II IL-4R deficient basophils IL-4 signaling is impaired at low ligand concentration. Thus, mast cell and basophil sensitivity to IL-4/IL-13 is different and in mast cells, lack of IL-13Ra1 expression likely explains their unresponsiveness to IL-13.

Supported by grants from Academy of Finland (grants: 25013080481 and 25013142041), The Competitive State Research Financing of the Expert Responsibility Area of Fimlab Laboratories (grant: X51409), Tampere University Hospital Support Foundation (grant: MK247), Tampere Tuberculosis Foundation, Nordlab Laboratories (grant: X3710-KT0011) and Orion Research Foundation.