bluuebunny/crossref_metadata_2025 huggingface dataset:
Using immune cell type-specific gene backgrounds for refined GO enrichment analysis: an example from CD4 T cell transcriptomics of TB-infected individuals
Authors: Pomaznoy Mikhail , Ha Brendan , Peters Bjoern
Published: January 2023

Gene Set Enrichment Analysis (GSEA) and its special case of GO enrichment analysis is a common step in biologically interpretation of gene lists resulting from –omics experiments. A known but under-appreciated aspect of any GSEA is the use of a background gene list, e.g. by discarding genes generally not expressed in the investigated cell type. We recently developed a web-application for interactive GO analysis of gene/protein signatures named GOnet (https://tools.dice-database.org/GOnet/). Within its framework we utilized DICE-DB (https://dice-database.org/) expression data of 15 blood cell types to allow analyzing gene signatures versus genes expressed in these cell types. To validate our approach, we applied GOnet to analyze a 74-gene transcriptomic signature of latent TB infection obtained by RNA-seq of CD4 memory T cells (Burel et al, 2018). Limiting the analysis to the background of genes expressed in CD4 T cells introduces significant differences in GO enrichment analysis results. Firstly, the category of “regulation of immunological synapse formation” is no longer enriched. As immunological synapse genes are very specific for T cells, without background correction this category can very easily seem ‘enriched’. Secondly, a more specific background renders a category of “positive regulation of MAPK cascade” significant. The latter category highlights important regulators contained in the signature. Overall we believe that using baseline gene sets based on immune cell types expression profiles is an important advancement to regular GO enrichment analysis in immunology studies, and by making relevant backgrounds readily available in GOnet, applying them becomes easier for immunologists.
Detection of small particles by flow cytometry: Analysis of small particle beads and extracellular vesicles
Authors: Jachimowicz Lauren A , Ye Peifang , Lu Yan , Guenther Garret , Nancy LI
Published: January 2023

The importance of cell-derived small particles has recently been recognized in multiple biological processes and is an increasing topic of investigation in biomedical research. Extracellular vesicles (EV) are cell-derived membranous vesicles that include exosomes, microvesicles, and apoptotic bodies. Small particle analysis by flow cytometry can bring much needed discoveries in small particle research, with the capability to detect rare events in significant numbers as well as measure surface protein levels. However, traditional flow cytometers were not capable of resolving them due to their small diameter and variable composition.

The aim of this investigation is to determine the capacities of the NovoCyte QuanteonTM flow cytometer to resolve small particles, using both beads and cell-derived EV. We first performed analysis of three commercially available small particle bead mixes ranging in size of 100nm-1300nm and determined the Quanteon is able to resolve beads as small as 100nm. Since beads scatter more light than similar sized membranous particles, it is important to measure biological samples to test the detection limits of the Quanteon. Therefore, HEK293 derived GFP lipoparticles were analyzed for scatter resolution and GFP expression. In addition, plasma was analyzed for purified EV with and without ADP addition, a molecule known to induce platelet activation and EV release. Next, EV from a cancer cell line, SW480, was analyzed for known EV markers CD9, CD81, and AnnexinV. The Quanteon was able to effectively analyze lipoparticles and secreted EV. In conclusion, the NovoCyte Quanteon equipped with new technologies allows better discrimination of small particles and provide increased capacity for research into EV.
Combining TruCulture and OptiMAP to Profile Human Immune Immunity
Authors: Hwang Shen-An , Eisinger Dominic , LaBrie Samuel
Published: January 2023

TruCulture® is a whole blood collection and culture system designed for use at clinical sites. TruCulture outperforms traditional peripheral blood mononuclear cell (PBMC) culturing methods, with improved reproducibility and higher levels of stimulation (Duffy et. al. Clinical Immunology 2017). The TruCulture proprietary culture medium ensures minimal non-specific immune activation, delivering a steady baseline and decreased variation during stimulating parameters. OptiMAP is a multiplex immunoassay panel that measures 13 cytokines of the major immune response pathways (TH1, TH2, TH17, and monocyte/macrophage). Using TruCulture and OptiMAP, we analyzed healthy donor whole blood responses to common stimulants: T cell activators, TLR agonists, and superantigens. The subjects ranged from 24 to 70 years old and consisted of 48% males and 52% females. The results indicated that healthy donors responded similarly to stimulation with anti-CD3/CD28 and Staphylococcal enterotoxin type B (SEB) in production of T cell cytokines (IFN-γ, IL-2, and IL-17). Response to SEB is positively correlated to the monocyte population in whole blood. T cell responses to SEB and CytoStim (an antibody based alternative to SEB) are similar in production of IFN-γ and IL-17, but CytoStim had a reduced stimulation of IL-2. All healthy donors responded to a TLR-4 agonist (LPS) by producing high levels of TNF-α, IL- 6, and IL-1β. LPS stimulated production of IFN-γ was similar to SEB, most likely due to bystander activation. The TLR-7 agonist (gardiquimod, GDQ) stimulated IL-10 levels at a level comparable to LPS. These data illustrate the utility of combining TruCulture and OptiMAP to investigate human immune stimulation pathways for clinical research.
Novel Flow-Based Multiplex Assay Panel for Quantifying Human Neuroinflammation Biomarkers
Authors: Sun Binggang , Chow Kay , Zhao Amy , Lehmann Jason , Jiang Weiping , Ji Shaoquan
Published: January 2023

The inflammation of neuronal tissues is triggered by various stimuli such as traumatic brain injury, stroke, protein aggregation and pathogens. Neuroinflammation cues such as brain injury lead to protein release and microglia activation, which in turn activate glial cells. Activated glial cells produce both pro- and anti-inflammatory factors to regulate cell migration, growth, tissue repair or cell death. Neuroinflammation is involved in acute brain injury, stroke, and many neurodegenerative diseases, such as Alzheimers’s and Parkinson’s diseases, as well as disruption and permeability of the blood-brain barrier. Measurement of the cytokines, chemokines, growth factors and other neuronal disease biomarkers is essential in understanding the mechanisms of neuronal disease progression, but this is limited by the small sample size of CSF. It is therefore desirable to be able to measure a panel of important neuroinflammation biomarkers simultaneously.

We have developed an assay panel targeting neuroinflammation biomarkers using fluorescence-encoded beads that are suitable for use on common lab flow cytometers. It allows for simultaneous quantification of the 13 key targets involved in neuroinflammation including VILIP-1, BDNF, free active TGF-β1, VEGF, IL-6, sTREM-1, β-NGF, IL-18, TNF-α, soluble RAGE, and CX3CL1 (Fractialkine). This panel has been developed with superior assay performance and specificity and has been validated by detecting expected values in biological samples including CSF samples from healthy and disease individuals. This high quality, low cost and easy to use panel will be a useful tool to the neuroinflammation research community.
Formal representation of immunology related data with ontologies
Authors: Vita Randi J , Overton James A , Cheung Kei-Hoi , Dunn Patrick , Burel Julie , Chan Syed Ahmad , Diehl Alexander D , Kleinstein Steven H , Sette Alessandro , Peters Bjoern
Published: January 2023

The Human Immunology Project Consortium (HIPC) is a multicenter collaboration between research centers performing large-scale human immunology studies that focus on profiling the human immune response to natural infection and vaccination. “Immune exposures” are events such as natural infection and vaccination whereby the immune system may or may not respond to the exposure. Many of the HIPC studies investigate the response of specific cell populations after a variety of immune exposures. In order to cross-compare results from the many different centers and projects, we established a standardized representation of immune exposures and cell descriptions that simplifies data collection. By standardizing how this data is collected and stored, the vast amount of data collected by these diverse projects is made significantly more useful and interoperable. The data collected by the HIPC projects is stored in the National Institute of Health, Division of Allergy, Immunology and Transplantation funded resource, the Immunology Database and Analysis Portal (ImmPort). ImmPort was modified to provide the necessary structured data fields to capture our standardized representation of immune exposures and studied cell populations with a set of data fields that primarily utilize formal ontology terms. We will discuss the process of modeling immune exposures and cell populations via ontology terms, including real life scenarios from HIPC projects, as well as collaborations with existing ontology projects in order to meet the specific needs of immunologists.
Development and application of an in vitro dendritic cell internalization assay to assess immunogenicity risk
Authors: Halimani Mahantappa , Atkuri Kondala , Goiberg Polina , You Zhiping , Steeno Greg , Tourdot Sophie , Xue Li , Lepsy Chris , Hickling Tim , King Lindsay
Published: January 2023

The objective of this research was to develop an in vitro dendritic cell based assay to assess if differences in internalization of biotherapeutic drugs (BioTx) and/or intra-cellular trafficking could be measured and used to help predict clinical immunogenicity risk. An unwanted immune response can adversely impact pharmacokinetics, safety and efficacy. Since immunogenicity in preclinical species is not clinically predictive there is a strong need for in vitro tools to enable BioTx drug design. Immature dendritic cells (iDCs) were selected because of their high fluid phase pinocytosis rates, ability to recycle IgGs and route immunogenic molecules to MHC-II compartments. Initial assay development evaluated iDC differentiated from a monocytic human cell line vs primary monocytes from fresh whole blood using IL4 and GM-CSF. Fluid phase internalization rates of Ovalbumin and Lucifer yellow decreased from day 3 to day 5 and CD80, CD86 and HLA-DR expression increased consistent with DC maturation. Imaging cytometry was used to measure internalization extent, rate and LAMP-1 co-localization of fluorescently conjugated ovalbumin and BioTx with known clinical immunogenicity incidence. Primary monocytes derived iDC were selected based on extent of internalization for further assay characterization in multiple donors. No target expression was observed. The extent of Adalimumab, Traztuzumab, and Utomilumab internalization was correlated with clinical immunogenicity incidence suggesting the assay may be able to detect frame work differences. Statistical power calculation of fold change set required donor numbers at 10. The assay can be used in combination with other in vitro immunogenicity assays to support BioTx design.
Imaging Cancer Immunology: Tracking Immune Cells in vivo with Magnetic Particle Imaging
Authors: Gaudet Jeff , Mansfield Jim , Goodwill Patrick
Published: January 2023

The rapid growth of research into immuno-oncology has fueled a need to be able to determine the location of a variety of immune cells in solid tumors. However, existing methods for that have been insufficient. Magnetic Particle Imaging (MPI) is a novel pre-clinical cellular imaging technique that can be used to non-invasively track iron-oxide tagged immune cells in vivo. In this study, we imaged the differences in biodistribution and migration of macrophages in naïve and tumor bearing mice. Imaging was performed with the MOMENTUM MPI system (Magnetic Insight Inc., CA). Macrophages were pre-loaded with tracers in vitro for tracking specific populations or loaded in situ for detection of localized inflammation. Initial macrophage biodistribution varied depending on the route of administration. Intravenous murine tail vein administration provided the best systemic delivery, but also resulted in macrophage accumulation within the liver and spleen. Direct intracranial injection increased local retention but resulted in the most tissue disruption. In the tumor bearing animals, macrophages were predominately detected in the expanding margins of the tumor. By combining the accurate quantification and specificity of MPI, information can be obtained on macrophage function and biodistribution. Other cells can also be labeled (dendritic cells, MDSCs, etc) and this information can be utilized to better understand the factors influencing immune cell migration in and around tumors.
A robust human immunophenotyping workflow using CyTOF technology coupled with Maxpar Pathsetter, an automated data analysis software
Authors: Li Stephen K.H. , Majonis Daniel , Bagwell C. Bruce , Hunsberger Benjamin C. , Baranov Vladimir , Ornatsky Olga
Published: January 2023

Mass cytometry, which utilizes CyTOF® technology, is a single-cell analysis platform that uses metal-tagged antibodies. An advantage of CyTOF is its ability to resolve more than 40 parameters in a single panel without the need for compensation, making mass cytometry an ideal solution for routine enumeration of immune cells. Developing a robust and highly multiplexed assay requires an optimized panel and analysis pipeline, and the analysis of complex panels is time-consuming and difficult to interpret without expertise in immunology.

The Maxpar® Direct Immune Profiling Assay™ coupled with Maxpar Pathsetter™ software is a sample-to-answer solution for human immune profiling using mass cytometry. The Maxpar Direct Immune Profiling Assay kit includes an optimized 30-marker panel contained in a lyophilized single-tube format, protocols for human whole blood and PBMC staining, and data acquisition instructions on a Helios™ system. Maxpar Pathsetter is an automated software that accepts FCS 3.0 files and reports cell counts, percentage calculations, and staining intensity. It also produces graphical elements such as histograms, dot plots, and a Cen-se’™ (t-SNE variant) graph for 36 immune cell populations.

We present analytical validation data on repeatability and reproducibility for the assay and on the precision and accuracy of Maxpar Pathsetter software. We demonstrate comparative performance between the lyophilized panel and a liquid panel of the same antibodies. Coupling Maxpar Pathsetter software with the Maxpar Direct Immune Profiling Assay reduces variability in sample preparation and subjectivity in data analysis. It allows researchers to have a streamlined solution for broad immune profiling using mass cytometry.
‘Evidence of improved transduction and preservation of relevant T cell subpopulations using different media conditions: Towards a novel CAR-T media formulation’
Authors: Martinez Becerra Francisco J , O’Connor Roddy , Ghassemi Saba , Heo David , Master Alyssa
Published: January 2023

The field of cell therapy is shifting away from animal origin components used in the culture or preservation of cells and replacing them with human or chemically defined components. However, this shift often leads to diminished cell proliferation and functionality due to the lack of physiologically relevant proteins. Therefore, there is an important need for generation of media supplements to optimize cell therapy. PhysiologixTM XF Human Growth Factor Concentrate (hGFC) is a cGMP, xeno-free media supplement that can be used as a replacement to supplements such as human serum. In this study, T cells were isolated from blood and cultured for 14 days in OpTmizer or X-VIVO 15 supplemented with 5% human serum or 2% PhysiologixTM XF. Population doublings and cell size were defined, as well as CD4/CD8 and memory markers (CCR7 and CD45RO) to assess phenotype. We detected an increase in naïve and central memory populations in 2% PhysiologixTM XF compared to human serum across all tested basal media. These populations have been described as highly relevant for the outcome of patients undergoing CAR-T cell therapy. We compared the transduction efficiency of a lentiviral-GFP in OpTmizer, X-VIVO 15 or RPMI, supplemented with 2% PhysiologixTM XF or 5% human serum at different multiplicities of infection (MOIs). Higher transduction efficiency was observed when using 2% PhysiologixTM XF at a high MOI of 4 and was conserved across all dilutions including a MOI of <1. These results indicate that PhysiologixTM XF can be used to optimize the generation of relevant T cell subpopulations and maximize transduction efficiency even at a low MOI, thus increasing the efficiency of vector usage. These results may lead to better clinical efficacy while reducing overall costs.
Improving expression estimates of HLA alleles in NGS data using personalized genomic sequences and its effect on eQTL mapping
Authors: Crinklaw Austin , Mahajan Swapnil , Pomaznoy Mikhail , Sette Alessandro , Vijayanand Pandurangan , Peters Bjoern
Published: January 2023

HLA genes are the most polymorphic in the human genome. Specific genetic variants in the HLA locus are frequently associated with diseases and other traits in GWAS studies. How much of those associations are due to functional differences in the peptide binding specificity of the various HLA genes encoded in the locus, vs. how many associations are due to differences in the expression level of the HLA genes has not been systematically determined. One hurdle in assessing HLA gene expression from RNA-Seq data when using standard analysis methods is the use of a single reference genome. We compared the estimated expression of HLA genes from 15 immune cell types of 91 individuals from the DICE database, using either the reference human genome sequence, or a personalized genome sequences based on HLA typing of the donors. This analysis showed that quantifying HLA gene expression using personalized genomic sequences allows to separate SNPs in association with HLA expression that are artifacts based on differences in read-mappability from other SNPs that are truly in association with personalized expression levels.
Comparative analysis of immunoglobulin IGHD genes in vertebrates
Authors: Bhardwaj Vinnu , Safonova Yana , Franceschetti Massimo , Rao Ramesh , Pevzner Pavel
Published: January 2023

Finding germline immunoglobulin (Ig) V, D, and J genes is a preliminary step in many antibody studies, including development of monoclonal antibody drugs. Recent studies show that the IMGT database, the state-of-the-art database of germline Ig genes, is incomplete for many species. Moreover, modern pharmaceutical studies often involve species not present in the IMGT database (e.g., hamsters, llamas). While V and J genes are conservative and can be inferred by homology, D genes among species are highly diverse. As modern sequencing technologies (Rep-Seq) have enabled scanning millions of antibody RNAs from a single organism, we developed a tool, MINING-D, for de novo inference of functional D genes using Rep-Seq data.

We applied MINING-D to 261 publicly available Rep-Seq datasets: 160 human and 101 non-human datasets, including mouse, rat, rabbit, camel, and rhesus macaque species. Most known D genes, their known and novel variations, and some D genes not presented in the IMGT database were inferred. We further validated a significant number of the novel genes and variations using genomic data.

Thus, our tool circumvents the need of an existing database and infers the functional D genes directly from expressed Ig RNAs. It can potentially speed up the process of antibody sequencing and design of antibody drugs for species with unknown genes. Results from multiple datasets enabled comparative analysis of D genes. For example, while the same germline D genes contribute to the diversity of antibody repertoires in healthy humans, only few of them are used in specific repertoires (e.g., hepatitis) and thus can be critical for recognition of their antigens. We suggest that the obtained results will help in disease monitoring and vaccine design.
Immunophenotyping extracellular vesicles using the CellStream flow cytometer
Authors: Pugsley Haley Renee , Davidson Bryan R , Morrissey Philip
Published: January 2023

Extracellular vesicles are membrane derived structures that include exosomes, microvesicles and apoptotic bodies. The importance of extracellular vesicles as key mediators of intercellular communication is not well understood. Exosomes have been shown to transfer molecules between cells, potentially transmitting signals. Exosomes are released under normal physiological conditions; however, they are also believed to serve as mediators in the pathogenesis of neurological, vascular, haematological and autoimmune diseases as well as cancer. Quantifying and characterizing extracellular vesicles in a reproducible and reliable manner is challenging due to their small size (exosomes range from 30 to 100 nm in diameter). Extracellular vesicle analysis can be done using high-magnification microscopy; however, this technique has a very low throughput. Attempts to analyze extracellular vesicles using traditional PMT based flow cytometers has been hampered by the limit of detection of such small particles and their low refractive index. To overcome these limitations, we have employed the Amnis CellStream that has the advantage of high throughput flow cytometry with higher sensitivity to small particles due to the CCD based, time-delay-integration image capturing system. Data will be presented using the CellStream flow cytometer to immunophenotype extracellular vesicles derived from red blood cells and platelets.
A novel method for high throughput TCR single cell VDJ-pairing with phenotypic analysis
Authors: Byrne-Steele Miranda , Pan Wenjing , Brown Brittany E , Hou Xiaohong , Depinet Mollye , Eisenhower Mary , Weber Daniel , Han Jian
Published: January 2023

Single cell transcriptional analysis reveals a wealth of information that is obscured when analyzing the RNA of a sample en masse. Furthermore, it is important to know the identity of the TCR chain VDJ-rearrangement associated with the phenotypic state. This information can provide direct calculations of clonal frequency in various cell subsets, tracking of specific lymphocytes with treatment, and reveal paired information for both chains of the receptor for downstream Car-T development. Here, we discuss a novel method that identifies paired VDJ-rearrangements for both TCR alpha and beta chains from single cells barcoded via the BD Rhapsody Express single cell system. The phenotype of the cell is obtained using the BD Rhapsody RNA-seq kit, while the receptor information is amplified from the same cDNA using an iRepertoire-derived method incorporating a multiplex mix of primers associated with both the TCR alpha and beta loci. This targeted-seq strategy requires less read depth when compared to template-switch approaches. In one experiment, 3,000 TCR-beta unique CDR3 (uCDR3) were identified from ~7,000 barcoded cells using 1.5 million NGS reads with a 0.83 Pearson correlation upon repeat. Approximately 1,000 TCR alpha-beta paired uCDR3 were also called from the data. Additionally, the BD phenotyping results independently verify the immune repertoire data since the TCR beta chain constant gene overlays with the TCR-beta uCDR3 clonotypes for CD4+ and CD8+ subsets but is largely absent in the CD14+ monocyte population for both analyses. Thus, the developed technology provides the ability to simultaneously pair both alpha and beta chains for thousands of T-cells while obtaining information related to the functional status of each cell.
Development of high-throughput methods used to identify and characterize novel interactions within the human secretome with focus on the Ig and TNF receptor superfamilies
Authors: Garrett-Thomson Sarah C , Almo Steven
Published: January 2023

While it is estimated that roughly 15% of the human genome encodes secreted proteins, many remain “orphans” with no known ligand or are poorly characterized. Even for those with known ligands, new binding partners continue to be identified that fundamentally reshape our understanding of their role in specific biological processes and diseases (i.e. PD-L1:B7-1, ICOS-L:CD28, PD-L2:RGMb). This highlights the need for systematic screening efforts to identify such interactions, broadening our general knowledge while also providing potential new targets for therapeutic intervention. To this end we have developed a set of novel technologies using both cell microarray and high-throughput flow cytometry techniques to screen for novel extracellular protein: protein interactions. Initially we focused on screening for interactions within the Ig and TNF receptor superfamilies, two protein families that are the central underpinning of our immune response but for which much is still unknown. We have generated two expression-validated libraries for cell-based screening, a 366 member Type I expression library of Ig and TNF receptor proteins and a ~3800 member full-length human plasma membrane protein library encompassing much of the human membrane proteome. Through rounds of screening we identified novel interactions for B7-1 (CD80), ICOS-L, TrkA and TrkC. The identified interactions were validated biochemically and are being characterized for their biological significance. Additionally we are using these technologies to characterize the binding interfaces of both known and newly discovered protein complexes as well as to screen for potential off-target interactions of protein-based therapeutics.
Alpha-1-Antitrypsin Ameliorates Diffuse Alveolar Hemorrhage in the Pristane Induced Lupus Model
Authors: Elshikha Ahmed Samir , Abboud Georges , Danos Abigail , Chen Mong-jen , Yuan Ye , Zeumer Leilani , Morel Laurence , Song Sihong
Published: January 2023

Diffuse alveolar hemorrhage (DAH) is a fatal complication of lupus with >50% mortality in humans. DAH can be induced in B6 mice by a single intraperitoneal (i.p.) injection of pristane. Human alpha-1-antitrypsin (hAAT) is an anti-inflammatory and immuno-regulatory protein. In this study, we investigated the protective effect of treatment with hAAT against pristane induced DAH development in B6, as well as in AAT-deficient (AAT-Ko) and hAAT transgenic (hAAT-Tg) mice. First, we characterized activated splenocytes from B6 and hAAT-Tg mice. We found that TNF-α production was significantly lower in the B cells, CD4+ and CD8+ T cells from hAAT-Tg mice as compared to B6 mice. Conversely, the frequency of CD4+CD25+ and CD4+CD25−IL-10+cells was significantly higher in hAAT-Tg than that in B6 mice. To test the effect of hAAT on DAH development, we treated B6 mice with or without hAAT (2mg/mouse/2 d) and induced DAH by a pristane injection. Two weeks after induction, control B6 mice showed severe lung hemorrhage, while hAAT treatment prevented lung hemorrhage. To investigate the mechanisms responsible for protection, we induced DAH in B6, AAT-Ko and hAAT-Tg mice. One week after induction, the frequencies of pro-inflammatory CD11b−Ly6Chi, pDCA-1+CD11clo, CD11c+CD11b+ and neutrophils were significantly lower in the peritoneal exudate cells of hAAT-Tg mice as compared to B6 and AAT-Ko mice. Moreover, the frequency of CD11b+Ly6Clow cells were significantly increased in hAAT-Tg. These results showed for the first time that hAAT has a therapeutic potential for the treatment of DAH by modulating myeloid cell phenotypes.
De novo fatty acid synthesis induced Sjögren’s syndrome by promoting Th17 differentiation
Authors: Luo Jing , Yin Zhinan
Published: January 2023

Primary Sjögren’s syndrome (pSS) is a chronic inflammatory autoimmune characterized by inflammatory infiltration of immune cells within lacrimal and salivary glands (SGs), which leads to the dysfunction of these exocrine glands. Th17 cells and their secreted cytokines play an important role in the pathogenesis of Sjogren’s syndrome, but the mechanism is still unclear. Recent clinical studies have found that metabolic disorders may be closely related to Sjogren’s syndrome. Our studies have also found that FASN, the key enzyme of lipid synthesis and metabolism in peripheral blood of Sjogren’s syndrome patients, is significantly elevated, and mainly expressed in Th17 cells. Fatty acid synthase (FASN) is downstream of ACC, and previous studies have shown that FASN activity influences both cancer and inflammation. However, it remains to be determined whether FASN is a viable target for Sjögren’s syndrome. Here, we demonstrate that FASN is a critical metabolic induced Sjögren’s syndrome by promoting the generation of inflammatory subsets of Th17 cells. In vivo, knocking out of FASN in Th17 cells, leads to reduction of Sjögren’s syndrome. These study demonstrate that FASN-mediated metabolism disorder of fatty acid synthesis may be a new mechanism to regulate Th17 cell function and lead to Sjogren’s syndrome, which can provide new directions and ideas for the treatment of Sjogren’s syndrome.
A rationally engineered DNase1-Fc fusion protein ameliorates autoimmune glomerulonephritis
Authors: Mouchess Maria , Suto Eric , Chan Sara , Dai Bingbing , Tam Christine , Cao Yi , Darwish Martine , He Dongping , Haller Susan , Eastham-Anderson Jeffrey , Franke Yvonne , Blanchette Craig , Lee Wyne , Austin Cary , Lazarus Robert , Yi Tangsheng
Published: January 2023

Loss of B cell tolerance and generation of autoreactive anti-nuclear antibodies are hallmarks of systemic lupus erythematosus and lupus nephritis. The mechanism of anti-nuclear immunoglobulin accumulation and clearance in lupus nephritis pathogenesis remains largely uncharacterized. Here, we show that innate immune activation in the NZB/W F1 mouse model and human lupus nephritis biopsies rapidly reduces expression of DNase1 (also called DNase I) in renal cortex proximal tubular cells. Unexpectedly, kidney DNase1 levels were no longer affected by TLR ligand agonism upon depletion of macrophages, indicating that local macrophage activation is required for the downmodulation of DNase1 in kidney epithelial cells. To overcome the loss of endogenous DNase1, we rationally designed a hyperactive actin-resistant variant of DNase1 with improved catalytic activity and acceptable in vivo pharmacokinetics. The engineered DNase1-Fc fusion protein ameliorates nephritis in a murine model of lupus and reduces immune complex deposition/complement fixation. Our data suggest that the loss of renal DNase1 through TLR signaling or other innate immune activation impairs clearance of autoreactive anti-nuclear immune complex deposits in the kidney to promote nephritis progression. While autoantibodies and autoreactive B cell activation are central to lupus pathogenesis, we hypothesize that renal cortical epithelial cells expressing high levels of DNase1 serve as gatekeepers to prevent detrimental nuclear immune complex deposition. Our findings provide a therapeutic rationale for using an engineered hyperactive actin-resistant DNase1-Fc for lupus nephritis.
Validation of lanthionine synthetase C-like 2 (LANCL2) as a therapeutic target for systemic lupus erythematosus
Authors: Leber Andrew , Hontecillas Raquel , Zoccoli-Rodriguez Victoria , Bassaganya-Riera Josep
Published: January 2023

Lanthionine synthetase C-like 2 (LANCL2) is a membrane receptor with downstream effects on immunity and metabolism including the differentiation of regulatory CD4+ T cells. Its natural ligand, abscisic acid (ABA), has been shown to be decreased in autoimmune and inflammatory disease. Given the implication of the LANCL2 pathway in autoimmune disease and the progression of LANCL2-specific ligands, such as BT-11, into clinical stage development, we assessed the role of LANCL2 in systemic lupus erythematosus (SLE). In a TLR7/8 resiquimod-induced model of SLE, the loss of LANCL2 significantly decreases survival (P = 0.0008) relative to wild-type, resulting in less than 10% survival and a greater than 60% reduction relative to wild-type. Similarly, LANCL2−/− displayed greater serum anti-dsDNA antibodies and higher proteinuria. Within spleens, the loss of LANCL2 resulted in greater IL-6 and TNF, higher proportions of T follicular helper cells and lower ratios of regulatory CD4+ T cells relative to IFNγ producing cells. The greater inflammation was also mirrored in transcriptional changes at the splenic level indicating a greater susceptibility to severe disease. These results merit further exploration into the implication of LANCL2 signaling in human SLE and characterization of the therapeutic efficacy of LANCL2-specific agonists for future SLE development. Thus, LANCL2 is a novel promising therapeutic target for SLE.
Small Molecule Inhibitor suppresses Tfh cell development in germinal center and reduces the severity of inflammation in a collagen-induced arthritis mouse model
Authors: Zhang Xin , Bhatia Nitasha , Migliore Frank , Hellmers Linh , Olayemi Gbemi , Mcleod Cara , Zakem Jerald , Davis William , Quinet Robert
Published: January 2023

Background

Rheumatoidarthritis (RA) is a autoimmune disease characterized by the accumulation of inflammatory cells in the joints, leading to hyperproliferation of synovial cells and tissue destruction. Our previous data showed autoreactive B cells could be induced by T follicular helper (Tfh) cells to undergo clonal expansion with in germinal center (GC), and ultimately differentiate into autoantibody producing plasma cells in RA. Here, we further investigated the role of Tfh cells in RA pathogenesis and the therapeutic effect of a small molecule inhibitor targeting Tfh cells (SMI-Tfh) in collagen-induced arthritis (CIA)mouse model.

Methods

CIA model was induced by immunization with chicken type II collagen in DBA/1 mice. Disease progression was monitored daily by the paw swollenness. Following the onset of clinical arthritis, mice were treated with SMI-Tfh. At the end of the study, blood, spleen, and affected paws were collected. Pathological changes were examined by H&E staining of tissue sections. Immunofluorescent histochemistry (IHC) staining and flow cytometry analysis were used to identify Tfh cells (CD4+CXCR5+ICOS+) in spleen and blood.

Results

Arthritis onset was developed at day 21 and with peak on day 42 after initial immunization. Destruction of articular cartilage, increased inflammatory cells and Tfh infiltration were observed in the synovial tissue. Mice treated with SMI-Tfh had significantly reduced Tfh cells in GC of the spleen (p<0.01) and less arthritis feature (p<0.05) in CIA mouse.

Conclusion

SMI-Tfh selectively inhibits GC-Tfh cells in the spleen and abrogates the severity of inflammatory arthritis. It may serve as a novel therapy by interfering with autoreactive GC-Tfh cells development in RA.
MyD88 and PKD1 play an essential role in the development of spontaneous polyarthritis in IL-1 receptor antagonist-deficient mice
Authors: Yoon Taewon , Cho Hongsik , Stuart John M , Yi Ae-Kyung
Published: January 2023

IL-1 is an important cytokine that is secreted by various cells, including synovial cells, and can induce the production of proinflammatory mediators. Due to the excessive and uncontrolled IL-1 signaling, IL-1R antagonist knockout mice (IL-1rnKO) develop spontaneous polyarthritis that mimics some pathogenic features of human rheumatoid arthritis (RA). MyD88 is a key signaling adaptor molecule for toll-like receptors (TLRs) and IL-1R. We have previously identified protein kinase D1 (PKD1) to play an indispensable role in the MyD88-dependent proinflammatory response. MyD88 and PKD1 may therefore confer regulatory role on arthritis development in IL- 1rnKO mice. To investigate this, we examined the synovial cell, T cell, and B cell responses, and the incidence and severity of arthritis in IL-1rnKO mice that are deficient in MyD88 or PKD1. We found that MyD88 deficiency and PKD1 deficiency inhibited the development of polyarthritis as well as reduced the levels of synovial macrophages in IL-1rnKO. T cells isolated from lymph nodes expressed significantly reduced CCR6, CXCR3, IFNγ, and IL-17 in MyD88-deficient IL-1rnKO mice. BAFF-R and TACI levels in splenic marginal zone precursor cells (MZP) and maturation of MZP to marginal zone B cells were significantly reduced in MyD88-deficient IL- 1rnKO mice. We are currently investigating whether PKD1 also play a regulatory role in T cell and B cell responses during the pathogenic course of arthritis in IL-1rnKO mice. Taking together, our results suggest that MyD88 and PKD1 play a critical role in the pathogenesis of arthritis in IL-1rnKO. Further insights into PKD1 and its role in arthritis development will contribute to better understanding innate signaling in the development of RA.
Targeting Chemokine Receptors CCR6 and CXCR2 in A Murine Model Of IL-36a-Induced Pustular Psoriasis
Authors: Campbell James J , Ebsworth Karen , Ertl Linda , McMahon Jeffrey P , Wang Yu , Yau Simon , Mali Venkat R , Chhina Vicky , Kumamoto Alice , Liu Shirley , Dang Ton , Newland Dale , Charo Israel F , Zhang Penglie , Schall Thomas J , Singh Rajinder
Published: January 2023

Several types of psoriasiform dermatitis are associated with increased IL-36 cytokine activity in the skin. A rare but severe psoriasis-like disorder, generalized pustular psoriasis (GPP), is linked to loss-of-function mutations in the gene encoding IL-36RA, an important negative regulator of IL-36 signaling. To understand the effects of IL-36 dysregulation in a mouse model, we studied skin inflammation induced by intradermal injections of pre-activated IL-36α. This type of inflammation was characterized by markedly increased total skin thickness and epidermal thickness. The inflammation was accompanied by increased concentrations of CCR6 and CXCR2 ligand chemokines in the skin, and large accumulations of CD4 T cells, neutrophils, and inflammatory DC. The accumulated inflammatory subsets in IL-36α-treated skin all expressed either CCR6 or CXCR2 to some extent, prompting the hypothesis that increased CCR6 and CXCR2 ligand concentrations in IL-36α-treated skin might constitute a viable target for relieving inflammation in this model. We found that the orally bioavailable small molecule CCR6/CXCR2 antagonist CCX624 did indeed reverse inflammatory cell accumulation in skin, as well as decrease total skin thickness and epidermal thickness. CCX624 was effective in both prophylactic and therapeutic dosing regimens. CCX624 treatment was more effective than saturating doses of anti-TNFα or anti-IL17RA, which are used in the clinic for treating GPP as first or second line treatments (respectively). These findings put CCR6 and CXCR2 forward as novel targets for a mechanistically distinct therapeutic approach for inflammatory skin diseases involving dysregulated IL-36 signaling, such as GPP.
1,25-Dihydroxyvitamin D3 ameliorates collagen-induced arthritis via suppression of Th17 cells through miR-124 mediated inhibition of IL-6 signaling
Authors: Zheng Song Guo , Zhou Li , Wang Julie
Published: January 2023

Objectives

To explore the molecular mechanisms in which vitamin D (VD) regulates T cells, especially Th17 cells in collagen-induced arthritis (CIA).

Methods

DBA1/J mice induced for CIA were intraperitoneally treated with VD. CIA clinical symptoms and inflammatory responses including Th1/Th17/Tregs percentages were determined and compared. Mouse naïve CD4+ T cells transduced with miR-124 inhibitor or not were polarized to Th17 cells with or without VD. Subsequently, cellular differentiation and IL-6 signaling moleculars were analyzed.

Results

VD treatment significantly delayed CIA onset, decreased incidence and clinical scores of arthritis, downregulated serum IgG levels and ameliorated bone erosion. VD downregulated IL-17A production in CD4+ T cells while increased CD4+Foxp3+Nrp-1+ cells both in draining lymph nodes and synovial fluid in arthritic mice. VD inhibited Th17 cells differentiation in vivo and in vitro and potentially functioning directly on T cells to restrain Th17 cells through limiting IL-6R expression and its downstream signaling including STAT3 phosphorylation, while these effects were blocked when naïve CD4+ T cells were transduced with miR-124 inhibitor.

Conclusions

VD treatment ameliorates CIA via suppression of Th17 cells and enhancement of Tregs. miR-124-mediated inhibition of IL-6 signaling, provides a novel explanation for VD’s role on T cells in CIA mice or RA patients and suggests that VD may have treatment implications in rheumatoid arthritis.
Identifying and engineering TCR specificity against solid tumor neoantigens
Authors: Naradikian Martin Souren , Montero Leslie , Hall Samantha , Thota Rukman , Frentzen Angela , Bahmanof Milad , Sternberg Luise , Lane Jerome , Kosaloglu-Yalcin Zeynep , Lanka Manasa , Miller Aaron , Peters Bjoern , Cohen Ezra , Schoenberger Stephen
Published: January 2023

Neoantigens (NeoAg) offer attractive therapeutic targets for directing a patient’s immune response to the immunogenic subset of mutations expressed exclusively by their cancer cells. Despite the specificity with which NeoAg enable tumor recognition, the majority of approaches for their identification rely on purely predictive methods such as calculating the ability of mutated peptides to bind to a patient’s set of HLA molecules. These methods have met with limited success in revealing natural targets present on tumor cells. We have developed a novel HLA-agnostic functional approach to NeoAg identification which combines genomic sequencing with bioinformatic analysis to nominate mutations for subsequent functional analysis using patient’s own T cells in an effort to identify natural responses generated under physiologic conditions. Using this, we identified a missense mutation (V205I) in the ribosomal protein RPS2 that is recognized by CD8+ T cells from tumor-infiltrating lymphocytes (TIL) of a metastatic HPV16+ Head and Neck Squamous Cell Carcinoma lesion. We then performed adoptive cellular therapy (ACT) using either unseparated TIL or those enriched for RPS2 V205I-specific CD8+ T cells and found the latter to be superior in controlling outgrowth of tumor of a PDX cell line generated from this lesion in NSG mice. Finally, we used single-cell transcriptomics to isolate the genes encoding the RPS2-specific TCR and show that it recognizes the mutated peptide bound to HLA-B*07:02. These results demonstrate that high-affinity NeoAg-specific T cell responses can be identified in cancer patients, that ACT of these cells can control tumor growth, and that the relevant TCR can be isolated for use in TCR engineering-based immunotherapy.
Substitution of alpha aminobutyric acid for cysteine at class I MHC anchor positions can enhance recognition of cancer epitopes
Authors: Sachs Abraham E , Peters Bjoern , Sidney John , Sette Alessandro , Rosenberg Steven A , Robbins Paul F
Published: January 2023

We curated a list of 11 known and validated class I-restricted cancer neoepitopes and tumor antigens that contain cysteine, six of which were restricted by HLA-A*02:01. We synthesized native (Cys-peptide) and alpha aminobutyric acid substituted (AABA-peptide) minimal epitopes and carried out co-culture assays in which responses of relevant T cell populations to titered doses of the native and substituted peptides were compared. The results indicated that T cells reactive with 6 of the 6 HLA-A*02:01 epitopes recognized peptides containing AABA substitutions at putative MHC anchor positions-at significantly lower concentrations than their Cys-pep counterpart. For 4 of the 5 non-HLA-A*02:01 restricted epitopes, T cells recognized AABA-peptides at lower concentrations than their native counterparts, while for the remaining epitope, the Cys-peptide was recognized at lower concentration than its AABA-peptide counterpart. Three of the HLA-A*02:01 restricted peptides, PHLPP1, HIVEP2, and gp100 contained two cysteine residues, one a putative MHC anchor and the second a putative T cell contact position. Substitution of ABBA for the putative T cell contact residue in these peptides appeared to reduce their ability to stimulate relevant cells. Overall, for 9 of the 11 (82%) cysteine-containing peptides evaluated, substitution of AABA for cysteine resulted in 10-fold or greater decrease in the minimum concentration of peptide recognized by T cells. We conclude that substitution of cysteine with AABA at MHC anchor positions can significantly improve the sensitivity of neoantigen screening assays by offsetting impairment that is likely to be due to cysteine oxidation, an effect that was observed for five HLA class I restriction elements.
Integrated profiling of T cells and tumor cells demonstrates metabolic adaptation essential for melanoma cell therapy
Authors: Martinez-Paniagua Melisa A , Haymaker Cara , Adolacion Jay R T , An Xingyue , Bandey Irfan N , Creasy Caitlin , Geiger Tamar , Harel Michal , Robinson Jonathan , Nielsen Jens B , Amritkar Amit , Woodman Scott , Roysam Badrinath , Hwu Patrick , Bernatchez Chantale , Varadarajan Navin
Published: January 2023

T cell-based therapies have shown promising results, but adoptive T cell therapy (ACT) for solid tumors like melanoma treatment has met limited success. The goal of this study was to identify attributes of T cell fitness important for optimal anti-tumor efficacy. This was accomplished by integrated profiling of expanded tumor infiltrating lymphocytes, TILs (responders (CR) and progressive diseases (PD)) co-cultured overnight with their autologous primary tumor cells with the aid of a suite of single-cell, transcriptional, proteomic and functional assays to construct a genome-scale metabolic model of the metabolism of TILs and tumor cells in direct competition with each other. Sorted live TILs after co-culture were used to performed RNA-seq, and proteome-wide profiling by mass spectrometry. Gene enrichment of the CR TIL suggested an increased expression in genes involving processes such as glycolysis, hypoxia, adipogenesis, and mTORC1 signaling. PD tumors showed an increase for epithelial to mesenchymal transition (EMT) and glycolysis. Comparisons of transcripts and proteins in both CR and PD were tightly correlated (Spearman rank 0.765). Since patient-derived cell numbers were limited (<50,000), we utilized genome-scale metabolic models to infer relevant metabolic pathways by comparison to the human metabolic Atlas (HMR2). We observed high enrichment in fatty acid β-oxidation processes in different organelles related to CR TIL and tumor. Lastly, we examined if increasing fatty acid oxidation in TILs might enable their increased survival in metabolically replete environments. These results show that the fatty acid metabolism pathway is associated with the ACT efficacy, and it can contribute to improving treatment.