bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cyanobacterial toxins and components induce expression of pro-inflammatory cytokines/chemokines and secretion of soluble cell receptors in a 3D skin cell model
Authors: Augustine Swinburne A J , Eason Tarsha N , Robillard Michelle C 
Published: August 2023

Abstract
Exposure to cyanobacterial toxins has been linked to several diseases and disorders including skin rashes and damage to the liver and kidneys. Previous research has shown that Microcystin-LR, the most common and toxic microcystin in fresh water, induces the NLRP3 inflammasome in response to administration to mice by oral gavage. In this study, we used a Reconstructed Human Epidermis to investigate immune system responses to cyanobacterial exposure. This 3D tissue model consisting of normal, human-derived epidermal keratinocytes was exposed to Spirulina, anatoxin-contaminated lake water, and phycocyanin. The cell supernatant was probed with a multiplex of 38 cytokines/chemokines and 14 soluble receptors to elucidate the immunological response. Our results revealed that: while most proteins were down-regulated, a number were up-regulated; the expression of many proteins were highly correlated and clustered together; and across the case study datasets, multiple cytokines/chemokines (G-CSF, GRO, IL-6, IL-8, and MCP-1) and soluble cell receptors (sCD30, sEGFR, sTNFRII and sVEGFR1, sVEGFR2 and sVEGFR3) were highly expressed when exposed to the toxins. Our findings correspond with preliminarily histological results showing tissue damage when the skin cells were exposed to the cyanobacterial components. This work shows great promise in the ability to examine the immune response to toxic exposures and aids in the development of techniques useful for studying the impact of harmful algal blooms on public health. Future studies will focus on mapping the immune pathway in response to harmful algal bloom exposure and studying the roles of each of the expressed cytokines and soluble cell receptors in the inflammatory response.
This work is supported by the United States Environmental Protection Agency.
A combination of TLR9 and STING agonists induces potent neopeptide-specific T cell immunity and improves ICB efficacy in tumor models
Authors: Hioki Kou , Castro Eiro Melisa D , Li Ling , Wilmsen Merel E P , Kiernan Caoimhe H , Brouwers-Haspels Inge , van Meurs Marjan , de Vos Kevin , Wildemans Marlous , Zhao Manzhi , de Wit Harm , Grashof Dwin G B , van de Werken Harmen J G , Mueller Yvonne M , Schliehe Christopher , Ishii Ken J , Katsikis Peter D 
Published: August 2023

Abstract
Immunotherapies such as immune checkpoint blockade (ICB) therapies have been developed in the last years as a promising strategy for the treatment of cancer, however there remains a need to improve their efficacy. The most critical factor that affects the efficacy of ICB therapies, is the frequency of tumor mutations, the associated neoantigens generated and the T-cell response against them. Therefore, it is expected that neoantigen vaccinations would improve ICB therapy efficacy by boosting the neoantigen-specific T cell response. The aim of this study is to develop an effective vaccine using a combination of TLR9 and STING agonists together with synthetic long peptides to induce a potent neoantigen-specific cytotoxic CD8+ T cell response. We first analyzed the efficacy of the vaccine formulation containing 20-mer OVA peptide and found that mice immunized with OVA peptide together with the adjuvant combination induced potent antigen-specific T cell responses and are capable of controlling tumor growth and improving survival in B16-F10-OVA tumor bearing mice. Moreover, we found that the vaccine together with OVA peptide is able to synergize with anti-PD-1 treatment in controlling tumor growth. Finally, we examined the immunogenicity of the vaccine formulation with neopeptides identified from two different tumor models. The vaccine formulation induced potent neoantigen-specific T cell responses in vivo. Our findings demonstrate that the vaccine formulation using the combination of TLR9 and STING agonists induces potent T cell immunity against synthetic long peptides and can serve as a promising immunogenic neoantigen vaccine platform.
Supported by the Department of Immunology of the Erasmus MC the Dutch Cancer Society (KWF grant 12837) and in part by a grant from International Joint Usage/Research Center, the Institute of Medical Science, the University of Tokyo, and Japan Agency for Medical Research and Development (AMED).
Effects of targeted epigenetic modifications on T – cell reprogramming
Authors: Norman Allison R , Ward Grace Anne , Zebley Caitlin C , Youngblood Ben A 
Published: August 2023

Abstract
Over the last decade there have been huge advances in DNA sequencing technology, allowing cancer patients to undergo genomic diagnosis within days of admission. Building upon genomic sequencing, epigenomic profiling approaches of tumor and infiltrating lymphocytes inform on the stage of the disease and immune response, but interpretation of these data remains hindered by issues of heterogeneity. Epigenetic programs, such as DNA methylation, play a vital role in the regulation of CD8+ T – cell development and differentiation. Here, we will investigate the effects of targeted epigenetic mechanisms on the function of effector memory T – cells. Hypomethylating agents, such as Decitabine, have been extensively used in myeloid malignancies including Acute Myeloid Leukemia and Myelodysplastic Syndromes; however, the impacts on T – cells have not been well studied. We hypothesize that the utilization of these epigenetic modifications will alter the function and phenotypic characteristics of the effector memory T – cells. These results will lead to new approaches for reprograming effector memory T – cells via removal of repressive DNA methylation. The outcome of this study will provide novel insight into our understanding of the epigenetic regulation of T – cells function; especially, how DNA methylation programs instilled during differentiation could be modified to improve T – cells function.
Molecular mechanisms of PBX1 regulation of CD4+ T cell inflammatory phenotypes
Authors: Park Yuk Pheel , Roach Tracoyia , So Sujung , Choi Seung-Chul , Ostrov David A , Yang Young , Morel Laurence 
Published: August 2023

Abstract
The pre-B cell leukemia homeobox1 (Pbx1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, Pbx1-d that lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and SLE patients than healthy controls. We have shown that overexpression of Pbx1-d in CD4+ T cells impaired Treg cell homeostasis and expanded inflammatory CD4+ T cells. Here, we investigated the molecular mechanisms by which PBX1 regulates T cells. We showed that PBX1 is preferentially expressed in Treg cells over other CD4+ T cell subsets, and that it is negatively regulated by TCR activation. Furthermore, PBX1-D protein was less stable than the normal isoform, PBX1-B, in a pulse chase experiment. PBX1-B but not PBX1-D has two predictive ubiquitin binding sites, K292 and K293, located in the DNA binding domain. Mutation of these two sites reduced PBX1-B stability to a level similar to PBX1-D, suggesting that a loss of function in the lupus associated allele through the ubiquitin-proteasome dependent pathway. In addition, contrary to PBX1-B, PBX1-D is retained in the cytoplasm without the help of the MEIS or PREP1 co-factors, indicating different requirements for nuclear translocation. Overall, these findings suggest that multiple transcriptional and post-transcriptional mechanisms are responsible for PBX1-D loss of function and induction of CD4+ T cell inflammatory phenotypes in SLE.
This work was supported by National Institutes of Health Grants AI045050
ARYL HYDROCARBON RECEPTOR REGULATES GENES IN COLONIC EPITHELIAL CELLS TO MITIGATE COLITIS SEVERITY IN THE DSS-INDUCED MURINE MODEL
Authors: Saxena Archana , Mitchell Chandani , Staley Shanieka , Bogdon Raymond , Roark Kasie , Hailey Michele , Nagarkatti Mitzi , Nagarkatti Prakash , Busbee Brandon 
Published: August 2023

Abstract
Current treatment of ulcerative colitis (UC) leaves a clear unmet need to target the colonic epithelial cells (CECs). In our previous study we found that aryl hydrocarbon receptor (AhR) expression on CECs has a vital role during indole-3-carbinol (I3C)-mediated protection against colitis. However, the precise mechanism behind this remains unclear. We investigated how intestinal regulatory components were altered in the absence or presence of AhR in CECs during I3C treatment under colitis (mouse model) or colitis-like conditions (colonic organoid). We generated conditional AhR-CEC knockout mice (AV) and induced colitis using the dextran sodium sulfate (DSS) model. Our results showed that the AV mice lost the protective effects of I3C against colitis. AV mice had higher disease scores and increased inflammation compared to controls. I3C treated AV mice that had DSS induced colitis were not able to regulate the colitis-associated parameters, even though flow cytometry analysis revealed AV mice were still capable of increasing protective interleukin-22 (IL-22) production in the colon. These results prompted us to study the CEC-specific mechanism involved in I3C-mediated protection against colitis. Whole transcriptome analysis of RNA isolated from enriched CECs of experimental mice was performed and results showed significant altered expression of several microRNAs, mucins (muc2 and muc13), and tight junction proteins (Cld3 and Cld4) in AV mice when compared to controls. RT-PCR data and direct effects of I3C on CECs (immunostaining) using organoids validated these results. This data concluded that AhR-dependent signaling orchestrates I3C-mediated protection of colitis.
NIH 5P20GM103641
IFN-γ is Necessary but not Sufficient to mediate Activated CD4+ T cell-induced epithelial cell damage and death
Authors: Cruz Michelle A , Llyons Renae , Malhotra Riya , Meng Shanshan , Fuiji Satoru , Stappenbeck Thaddeus , Levine Alan D 
Published: August 2023

Abstract
Bidirectional communication between the intestinal epithelial barrier and the immune cells in the underlying lamina propria is critical to human health. This study investigates the impact of human T cell subtypes on intestinal epithelial cell phenotype and survival. Blood-derived CD4+ or CD8+ T cells were isolated from healthy human participants using negative magnetic separation and co-cultured with healthy human colonic epithelial stem cell spheroids suspended in Matrigel. A subset of T cells was activated by anti-CD3/CD28 cross-linking for 24 h prior to co-culture. Only activated CD4+ T cells (ACD4) caused significant epithelial cell damage and death as measured by histological scoring of brightfield microscopic images and zombie violet+ epithelial cells detected by flow cytometry.
The mechanism of cell damage and death was determined to be via soluble mediators, in that conditioned media from ACD4 but not activated CD8+ T cells (ACD8) significantly induced spheroid cell damage and death. A Luminex cytokine multiplex assay revealed that high levels of IFN-γ, IL-17, and TNF-α were produced by ACD4 as compared with ACD8. Neutralizing IFN-γ antibody completely protected spheroids from death induced by ACD4. In contrast, the addition of only IFN-γ protein did not induce spheroid cell death. This finding conflicts with currently published literature that indicates IFN-γ protein is able to induce spheroid cell death for both humans and mice. In addition, ACD8 also secrete IFN-γ, although to a lesser extent than ACD4, yet do not initiate spheroid cell death. These results indicate that there is a unique mixture of immune mediator released by ACD4s that mediate spheroid damage and death.
DP1 DA-037997
T32 GM007250
The roles of epidermal Blimp-1 in atopic dermatitis
Authors: Yang Hao-Wen , Wu Yen-Fei , Yang Shii-Yi , Lin Kuo-I 
Published: August 2023

Abstract
Keratinocytes are the main cell type constructing the epidermis of animals, which can further differentiate into corneocytes and express multiple innate immune receptors. B lymphocyte-induced maturation protein-1 (Blimp-1) plays a crucial rule in directing cornification of keratinocytes. We previously reported that keratinocyte-specific deletion of Prdm1, the gene encoding Blimp-1, induced spontaneous inflammation in the skin of adult mice. We further found that deletion of Prdm1 in keratinocytes in Balb/c genetic background caused hyper-production of type 2 cytokines and thymic stromal lymphopoietin (TSLP) in the skin, and staphylococci colonization, which are characteristics of atopic dermatitis (AD). Since K5-CreER+Prdm1f/f (CKO) mice in Balb/c background exhibited AD-like symptoms, we wondered whether they are more susceptible to the disease progression in a murine AD model induced by MC903, a vitamin D3 analog enhancing TSLP production by keratinocytes. We found that CKO mice showed more immune cell infiltration in ear skin, developed more severe epidermal hyperplasia, and induced more TSLP secretion in the skin following topical application of MC903. Recent studies showed that staphylococci colonization activated the transcription of endogenous retroviruses (ERVs) in murine skin, while excess ERV expression led to skin inflammation. As staphylococci colonized on our CKO mouse skin, we hypothesized that enhanced ERV expression may also link to skin inflammation of CKO mice. Indeed, both in vivo and in vitro deletion of Prdm1 in keratinocytes resulted in elevated levels of ERV transcripts. We are studying the contribution of elevated ERV transcripts in Blimp-1-reduction-mediated AD-like skin inflammation.
IgE production and stability regulated by Glycosylation in vivo
Authors: Roy Avishek , Meregini Steve , Chen Zhenglan , Nair-Gill Evan , Ludwig Sara , Russell Jamie , Quan Jiexia , Hildebrand Sara , Moresco Eva , Yue Tao , Beutler Bruce , SoRelle Jeffrey 
Published: August 2023

Abstract

Rationale
Essential functions of IgE have been attributed to specific glycosylations such as oligomannose (Fcer1a binding) and sialylation (pathogenicity). However, no study has examined how glycosylation affects IgE stability in vivo.

Methods
Forward genetic screening for IgE specific phenotypes was performed using N-ethyl-N-nitrosourea (ENU) mutagenized mice. One low IgE phenotype, named benadryl, linked to a mutation in Mpi, which is essential for N-linked glycosylation. Benadryl was validated by CRISPR knock-in . Immunological studies were performed to determine the causative mechanism (passive/ active anaphylaxis, mast cell analysis, conditional knockout in T, B and dendritic cells, antibody production in vivo and in vitro, using IgE-Venus, IgE-KO, and IL-4 GFP mice).

Results
Benadryl mice had low total and papain-specific IgE responses with resistance to active anaphylaxis. In vitro and in vivo mast cell activation studies resulted in no difference in response to passive sensitization anaphylaxis. Both, in vivo and in vitro cellular staining in the conditional knockout showed no decrease in IgE B cells. IgE from Mpi deficient mice was produced and secreted from B cell culture. When de-glycosylated IgE was injected to IgE KO mice, it was cleared 10-fold faster than native IgE.

Conclusions
Glycosylation status determines not only IgE functionality but in vivo stability. Mpi deficient mice produce IgE hypoglycosylated in nature and therefore it is cleared faster escaping anaphylaxis. Modulating IgE glycosylation represents a novel way to reduce IgE below pathogenic levels.

NA
AMBRA1 control immune-specific gene expression in T lymphocytes
Authors: Yao Yikun , Lenardo Michael 
Published: August 2023

Abstract
Autoimmune lymphoproliferative syndrome (ALPS) is a disease characterized by the dysregulation of cell death in lymphocytes, resulting in symptoms such as hepatomegaly, splenomegaly, and anemia. While most patients with ALPS have a mutation in the FAS gene or within the FAS pathway, which helps mediate cell death, some patients who present with ALPS-like symptoms do not have known causative gene mutations. To identify other genes that confer resistance to cell death, and thus may play a role in diseases such as ALPS, we performed a whole genome CRISPR screen of Fas-mediated cell death and identified AMBRA1, a protein previously studied in autophagy and E3 ubiquitin ligase activity. We verified the screen results and indeed found that AMBRA1 KO cells resist Fas-mediated cell death. To identify the mechanism of how AMBRA1 contributes to cell death resistance, we performed cycloheximide chase assays, qPCR, and proteomic analysis and confirmed that AMBRA1 regulates FAS expression. Furthermore, we found that AMBRA1 itself can be upregulated by TCR stimulation through the CD28-PI3K-mTORC1 pathway. Mass spectrometry analysis and subsequent experiments also confirmed that AMBRA1 regulates other immune genes. Together, these data establish a novel function for AMBRA1 as a critical regulator in T cells and unveil new insights into how AMBRA1 may contribute to ALPS and dysregulated cell death in other immune diseases. This research was supported by the Intramural Research Program of the NIH.
NK-92-CD16A cell externalization of CD107a for detection of antigen-specific antibodies able to support ADCC: sensitive discrimination of antibody a-fucosylation
Authors: Amaya Judith B Cruz , Walcheck Bruce , Smith-Gagen Julie , Lombardi Vincent , Hudig Dorothy 
Published: August 2023

Abstract
Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes kills cells infected with enveloped viruses. Antiviral ADCC has three components: antigen-specific IgG1 or IgG3; effector cells with CD16A that recognize the Fc of IgG; and antigenic proteins in the plasma membranes of infected cells. Afucosylated antibodies support ADCC better than fucosylated antibodies; afucosylated fractions vary among individuals. Current assays that detect afucosylated antibodies to specific viruses involve antigen-specific antibody purification and mass spectrometry. We are developing clinical bioassays to detect antibodies that will support ADCC and assess their extents of afucosylation. Our pilot assay uses externalization of CD107a by NK-92-CD16A cells, this’ occurs when NK cells recognize cell-bound antibodies. We used two humanized monoclonal antibodies on CD20, GA101 WT or glycoengineered GE, 10% or ~50% afucosylated, with Raji cells. Cellular CD107a externalization was more sensitive than AF-647 donkey anti-IgG to detect total cell-bound antibody. The effective concentrations (EC50s) of the two antibodies for CD107a externalization differed by &gt;15-fold. The more afucosylated GA101-GE was more detectable, at 0.2 ng/ml (=/&lt; 700 Abs/Raji cell). CD107a externalization increased with excess antibody-bearing cells. Short 37 °C incubation (40 minutes) optimized detection of ADCC over intrinsic NK activity. Controls of GA101-GE alone and 56 °C heated human serum lacked effects on CD107a externalization. The assay appears compatible with formaldehyde treatment before NK cell staining. This sensitive bioassay appears suitable for assess serum samples for antiviral antibodies that will support ADCC.
UNR Foundation C.E. Hudig Fund.
Cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) of peripheral blood to dissect the effect of different spondyloarthropathies
Authors: Gill Tejpal , Davin Sean , Ogle Kimberly , Ahuja Jasvinder , Choi Dongseok , Martin Tammy , Rosenbaum James T 
Published: August 2023

Abstract
Spondyloarthritis is a family of immune mediated inflammatory disorders with overlapping articular and extraarticular clinical manifestations and a strong association to human leukocyte antigen (HLA)-B27. Despite a strong association of HLA-B27 with prototypic spondyloarthropathies such as axial spondyloarthritis (axSpA) and acute anterior uveitis (AAU), the underlying mechanisms are not known. Here, we aim to determine whether HLA-B27 associated axSpA and AAU are associated with common or divergent immunological markers in the blood. Previously, we did single cell RNA-seq analysis on a small number of patients with AAU and axSpA, which revealed increase in T cell, MAIT cells and NK cell clusters in AAU patients, whereas axSpA patients had increase in MAIT cells, gd-T cells, ILCs and Tregs and decrease in naïve CD8 T cell clusters, suggesting that distinct signatures maybe associated with axSpA and AAU. Changes in ILCs or MAIT cells may suggest involvement of the gut mucosal surface in pathogenesis. This is consistent with our previous studies showing gut microbial perturbation in clinical samples and in rat model of spondyloarthritis.
Leveraging on our preliminary data, we are performing CITE-seq to capture paired transcriptome and cell surface epitope expression in individual PBMCs from 5 subjects each with axSpA, AAU and axSpA+AAU in comparison with healthy individuals (5 HLA-B27 negative and 5 HLA-B27 positive). Since, HLA-B27 allele is also present in healthy population, these analyses will allow us to dissect the effect of host genetics from the disease. These studies represent potential advances in the understanding of spondyloarthritis and may reveal genetic and disease biomarkers associated with axSpA and AAU.
TG (principal investigator) is a recipient of research awards from the Spondylitis Association of America, SPARTAN (Spondyloarthritis Research and Treatment Network), GRAPPA (Group for Research and Assessment of Psoriasis and Psoriatic Arthritis), The Collins Medical Trust, Medical Research Foundation of Oregon and, Innovation Award from OHSU. This work was funded in part by the Grandmaison Fund for Autoimmunity Research. JTR is a recipient of the Innovative Research Award, Rheumatology Research Foundation.
Regulation of respiratory CD8+ T cell immunity by suppressive monocyte-like dendritic cells
Authors: Oliva Kimberly , Reagin Katie , Klonowski Kimberly D 
Published: August 2023

Abstract
Active immune suppression can mediate the balance between protective cellular immunity and harmful immunopathology. This suppression can occur locally, at an infection site, or in regional draining lymph nodes (dLN). Immune regulation is of particular importance in sites such as the lung where aberrant immunopathology can result in loss of tissue function. We have recently identified a novel population of CD11b+CD103+CCR2+ monocyte-like dendritic cells (moDCs) which directly suppress CD8+ T cell proliferation in vitro. Respiratory infection with RNA viruses recruits these moDCs either exclusively to the dLN (after vesicular stomatitis virus infection) or both the dLN and site of viral replication (after influenza infection). Exclusive depletion of moDCs from the dLN using CCR2-DTR bone marrow chimeras results in enhanced respiratory CD8+ lung tissue resident memory cell formation. By contrast, depletion of moDCs from both the dLN and respiratory tract following influenza infection results in enhanced respiratory CD8+ T cell responses coupled with fatal immunopathology. Together, these data suggest that suppressive moDCs govern key aspects of respiratory immunity by regulating the function and development of long-lived CD8+ T cells, thereby balancing immunity and adverse pathology in the context of viral infection.
Supported by grant from NIH (R21 AI131093)