bluuebunny/crossref_metadata_2025 huggingface dataset:
CSF1R blockade in fructose-fed rhesus macaques improves clinical parameters of prediabetes.
Authors: Stahr Natalie , Phou Amy , Glover Colin , Perkins Jeanne , Haupt Erin , Palmer Clovis , Ardeshir Amir , Kim Woong-Ki
Published: November 2024

We have previously found that depletion of CSF1Rhi macrophages (Mθ) using a rhesus IgG1 recombinant antibody against CSF1R (α-CSF1R) reduced total cholesterol and improved insulin sensitivity in naturally prediabetic rhesus macaques (RM). High fructose diet (HFD)-fed RMs have been established as a model of metabolic syndrome, with increased insulin resistance (IR) and increased monocyte chemoattractant protein 1 (MCP-1). We hypothesize that depletion of CSF1Rhi tissue Mθ using a monoclonal α-CSF1R and replacement with freshly differentiated Mθ will alleviate inflammation in tissues and ameliorate prediabetic disease in HFD-fed RMs.

We induced IR and heightened hbA1C, indicating prediabetes in eight aged Indian RMs through administration of a HFD for 6 months. RMs were treated with either α-CSF1R or an IgG1 control administered intravenously (15 mg/kg) once every 2 weeks for 6 weeks. Clinical parameters such as plasma levels of total cholesterol, glucose, insulin, adiponectin, CSF1, IL1β, MCP-1 were monitored throughout treatment and up to 8 weeks after the 3rd injection. Mθ subsets were quantified by immunofluorescent (IF) staining.

Treatment with α-CSF1R resulted in decreased total cholesterol and insulin, increased adiponectin and CSF1. IF staining confirmed the depletion of CSF1Rhi macrophages in insulin sensitive tissues. Treatment with α-CSF1R resulted in improvement in multiple clinical parameters associated with prediabetes.
DUSP8 Promotes IL-9 Transcription by Inactivating Pur-α leading to asthma and atopic dermatitis
Authors: Chuang Huai-Chia , Tan Tse-Hua
Published: November 2024

Dual-specificity phosphatase 8 (DUSP8) is a member of the DUSP family. DUSP8 dephosphorylates and inactivates the kinase JNK. DUSP8 protein levels are predominantly expressed in CD4+ T cells and platelets. DUSP8 dephosphorylates and inactivates specifically JNK in Jurkat T cells; however, the in vivo role of DUSP8 in T cells remains unclear.

Using T-cell-specific DUSP8 conditional knockout (T-DUSP8 cKO, DUSP8f/f;CD4-Cre) mice, mass spectrometry, and chromatin-immunoprecipitation sequencing, we found that DUSP8 stimulated IL-9 gene expression and Th9 differentiation. TGF-β stimulated DUSP8 phosphatase activity in T cells. Mechanistically, DUSP8 dephosphorylated the transcriptional repressor Pur-α upon TGF-β signaling, leading to the nuclear export of Pur-α and subsequent IL-9 transcriptional activation. Furthermore, Il-9 mRNA levels in murine T cells were induced in Pur-α knockout. Reduction of IL-9 levels in T cells of T-DUSP8 cKO mice was reversed by Pur-α heterozygous knockout. Consistently, T-DUSP8 cKO mice displayed reduction of IL-9 and Th9-mediated immune responses in allergy, autoimmune responses, and anti-tumor immunity. Remarkably, DUSP8 overexpression and DUSP8-Pur-α interaction indeed occurred in the peripheral blood T cells of patients with asthma or atopic dermatitis, contributing to IL-9 overproduction in patients’ T cells. Thus, DUSP8 plays a critical role in the pathogenesis of asthma and atopic dermatitis, as well as autoimmune disease and cancer.
Requirement for Toll-like Receptor 4 (TLR4) in LPS-induced lung inflammation caused by atypical Cyanobacteria Lipopolysaccharide
Authors: Swanson-Mungerson Michelle , Monroy Del Toro Rosalinda , Incrocci Ryan , Williams Philip , Mayer Alejandro , Swartzendruber Julie
Published: November 2024

Cyanobacterial blooms found in freshwater sources cause multiple human diseases, including lung inflammation. Cyanobacteria produce atypical lipopolysaccharides (LPS) that can act as either weak TLR4 agonists or even antagonists in in vitro and in vivo models. However, our laboratory demonstrated that LPS from the cyanobacteria Geitlerinema sp. activates immune cells, despite containing altered LPS sugar side chains and inducing modified TLR4 signaling. Additionally, Geitlerinema sp. LPS induced lung inflammation in vivo based on significant neutrophil infiltration 24 hours post LPS exposure. In the current study, we extend our initial findings and asked whether the atypical LPS from an additional cyanobacterial strain, Brasilonema sp., also induces lung inflammation and if TLR4 is required. Our data demonstrate that in vitro, Brasilonema sp. LPS stimulates both Interleukin (IL)-6 and IL-8 production by immortalized airway epithelial cells that is TLR4-dependent, since the addition of the SPA-4 peptide blocks LPS-mediated production of these cytokines. More importantly, Brasilonema sp. LPS induced lung inflammation in wildtype mice but did not induce lung inflammation in TLR4-deficient mice, suggesting that the atypical LPS from cyanobacteria binds to TLR4 to induce lung inflammation. These findings help us to understand the impact of cyanobacteria LPS in neutrophil-dependent airway inflammation in individuals living near freshwater sources contaminated with cyanobacteria.
Food allergies as a promoter of transient anxiety-like behavior
Authors: Bustamante Hernandez Leslie
Published: November 2024

Food allergies impact human health, including mood. Allergen challenges in mice promote central amygdala activation, the integrative hub for anxiety and other mood disorders. Indeed, food allergic individuals show higher prevalence of anxiety. We hypothesize that food allergies induce anxiety. Female BALB/c mice received OVA/alum sensitization (or PBS/alum for control) subcutaneously on days 0 and 7, followed by five OVA oral challenges every three days from days 14 to 28. Behaviors were assessed using standard anxiety tests (open field test, OFT, elevated plus maze, EPM). Development of allergy was confirmed by increased specific IgE, anaphylaxis, and diarrhea. Allergic mice showed avoidance to open and elevated areas (EPM) when compared with the control group (p= 0.0425). We found that allergic animals showed less time spent in the center of the arena (p= 0.0008, OFT) compared with controls, suggesting the development of anxiety-like behavior. Because the locomotor activity (total distance and speed) was not different between allergic and controls, we confirmed a true anxiety-like phenotype induced by food allergy. Notably, we found that only one allergen challenge was enough to induce anxiety. However, this behavior was transient as there was no difference two weeks after the last allergen exposure. Our data suggest that this anxiety is dependent on allergen challenge and we propose that allergic inflammation might contribute to mental disorders.
The role of Per2 in atopic dermatitis experimental model
Authors: Kang Tae Jin
Published: November 2024

Atopic dermatitis (AD) is a chronic inflammatory skin disorder associated with defective skin barrier. The Clock gene Per2, one of circadian genes, has vital role in inflammation and it is up regulates inflammatory cytokines. In this study we studied the Per2 role in RAW 264.7 by transfecting with Per2 siRNA for knockdown specific Per2 gene and atopic dermatitis animal model using establishing DNCB induced Per2-/- mice. RAW 264.7 cells were simulated with LPS for 12 hours, then measured circadian and cytokine gene expressions. We found that PER2 induce the inflammation and enhance the expression of pro-inflammatory cytokines such as TNF-a and IL-6 by suppressing Bmal-1, which has anti-inflammatory activity, and PER2 is inducing nitric oxide (NO) level too. RAW 264.7 cells transfected with Per2 siRNA was not stimulated by LPS treatment and produced lower levels of inflammatory cytokines and NO by Bmal-1 up-regulation. We also examined activity of Per2 in DNCB induced atopic dermatitis animal model by using Per2+/+ and Per2-/- mice. We found Per2+/+ mice has higher number of Th2 cells, up-regulated Th2 cytokines and GATA3 expression, suppressed BMAL1 expression. Per2 -/- mice showed low levels of Th2 cytokines and IgE levels, less Th2 cells. We hypothesized PER2 has prominent role to induce inflammation and atopic dermatitis
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Authors: Srivastava Neetu , Hu Hao , Peterson Orion , Vomund Anthony , Stremska Marta , Zaman Mohammad , Giri Shilpi , Li Tiandao , Lichti Cheryl , Zakharov Pavel , Zhang Bo , Abumrad Nada , Chen Yi-Guang , Ravichandran Kodi , Unanue Emil , Wan Xiaoxiao
Published: November 2024

Autoimmune disorders are characterized by destruction of specific tissues through invasive, antigen-specific responses. Yet, the influence of intrinsic tissue properties on susceptibility to autoimmunity remains uncertain. This study explored whether the proinflammatory features of resident macrophages in pancreatic islets could create an environment conducive to diabetic autoimmunity. Our analysis revealed elevated expression of CXCL16, a chemokine and scavenger receptor for oxidized low-density lipoproteins (OxLDL), in islet macrophages from both mice and humans, irrespective of autoimmune predisposition. Deleting Cxcl16 in NOD mice significantly suppressed the development of T1D. Mechanistically, the loss of Cxcl16 significantly impaired the capacity of islet macrophages to take up OxLDL. This coincided with OxLDL accumulation in pancreatic islets, leading to a reduction in intra-islet transitory (Texint) CD8 T cells displaying proliferation and effector signatures. Ferroptosis contributed to the reduction of these intra-islet Texint CD8 T cells, which are susceptible to oxidative stress. PD-1 blockade, however, rescued this cell population in the islets and reversed diabetes resistance in Cxcl16-deficient mice. Therefore, OxLDL scavenging by islet macrophages contributes to the local differentiation of pathogenic CD8 T cells. These findings suggest that the normal tissue microenvironment can facilitate autoimmune pathogenesis in predisposed individuals.
Oxidative stress is a shared characteristic of ME/CFS and Long COVID
Authors: Shankar Vishnu , Wilhelmy Julie , Curtis Ellis , Michael Basil , Cervantes Layla , Mallajosyula Vamsee , Davis Ronald , Snyder Michael , Younis Shady , Robinson William , Shankar Sadasivan , Mischel Paul , Bonilla Hector , Davis Mark
Published: November 2024

More than 65 million individuals worldwide are estimated to have Long COVID (LC), a complex multisystemic condition, wherein patients of all ages report fatigue, post-exertional malaise, and other symptoms resembling myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). With no current treatments or reliable diagnostic markers, there is an urgent need to define the molecular underpinnings of these conditions. By studying bioenergetic characteristics of peripheral blood lymphocytes in over 16 healthy controls, 15 ME/CFS, and 15 LC donors, we find both ME/CFS and LC donors exhibit signs of elevated oxidative stress, relative to healthy controls, especially in the memory subset. Using a combination of flow cytometry, bulk RNA-seq analysis, mass spectrometry, and systems chemistry analysis, we observed gender-specific aberrations in ROS clearance and damage pathways. While ME/CFS females exhibit higher total ROS and mitochondrial calcium levels, ME/CFS males have normal ROS levels, but larger changes in lipid oxidative damage. The higher ROS levels correlate with hyperproliferation of T cells and can be attenuated by metformin, suggesting this FDA-approved drug may be an effective treatment. Thus, we report that both ME/CFS and LC are mechanistically related and can be diagnosed with quantitative blood cell measurements. We also suggest that effective, patient tailored drugs might be discovered using standard lymphocyte stimulation assays.
Pharmacological activation of LANCL2 provides immunometabolic support for the restoration of cognitive function markers in a mouse model of Alzheimer’s disease
Authors: Leber Andrew , Hontecillas Raquel , Tubau-Juni Nuria , Fitch Sarah , Bassaganya-Riera Josep
Published: November 2024

LANCL2 is a member of a three-protein family of receptors that acts as the mammalian receptor for abscisic acid (ABA). LANCL2 expression is significantly diminished both with the brains of 3xTg mice as well as human patients with Alzheimer’s disease. Immunologically, LANCL2 expression is closely associated with regulatory CD4+ T cells and their suppressive capacity as well as within microglia. Treatment of mice with ABA improved spatial memory and reduced amyloid beta load. To study the effects of LANCL2 activation in vivo, we created a novel agonist. After 12 weeks of daily oral treatment at 20 mg/kg, LANCL2 activation significantly reduced inflammatory immune cells including neutrophils, Th17 and Th1 cells in 3xTg mice relative to vehicle control. Further, treatment with LANCL2 agonists reduced the expression of key markers in the brain associated with impaired cognitive function to levels observed within age-matched, negative control WT mice. Activation of LANCL2 prevents the polarization of microglia into a reactive phenotype and supports the metabolic demands of amyloid beta phagocytosis. LANCL2 drugs can combat Alzheimer’s disease progression, given that reactive microglia polarization not only diminishes microglial phagocytic capacity but also increases the production of inflammatory cytokines. The LANCL2 pathway’s transformative potential merits development as a promising therapeutic mechanism for Alzheimer's disease and diverse age-related cognitive impairment.
Impact of monocytes on the proliferation of highly activated NK-like cells (GAIA-102)
Authors: Yuan Shuyue , Harada Yui , Morodomi Yosuke , Nagata Hiroko , Yonemitsu Yoshikazu
Published: November 2024

[Background] There has been a growing clinical application of therapies using autologous and allogeneic natural killer (NK) cells. Researchers across the field have been developing various methods to amplify NK cells, highlighting the increasing importance of NK cell amplification techniques. We have already developed a technique to amplify highly active NK-like cells (GAIA-102) from allogeneic peripheral blood mononuclear cells (PBMCs) with several cytokines, and currently conducting three clinical trials utilizing this technology. However, it remains unclear which components of PBMCs contribute to the proliferation of NK cells, a question that is central to further advancing this field.[Method] We utilized magnetic beads to deplete various blood cell components from PBMCs to investigate the effects of the presence or absence of each component on the cultivation of GAIA-102. We then evaluated the dynamics of cell proliferation and surface marker expression changes.[Results] The removal of specific components did not impact the cytotoxicity or the surface marker profile (ITGA1+/CXCR3+/CCR5+/CCR6+) of the original GAIA-102. Interestingly, eliminating monocytes from the culture markedly decreased GAIA-102 cell proliferation, a more substantial effect than when B cells were removed.[Conclusions] Our study suggests that monocytes may play a crucial role of GAIA-102 proliferation. Unraveling the impact of monocytes on this process will be a vital direction for future research.
Batf3 is a critical transcription factor in IL-2 mediated programming of CD8+ T cell memory.
Authors: Nagaraja Shashank , Pipkin Matthew
Published: November 2024

CD8+ T cell activation triggers chromatin remodeling and transcription factor (TF) binding, leading to differentiation into effector (TEFF) and a spectrum of memory (TMEM) phenotypes. Previous studies have found modified chromatin accessibility at loci encoding Basic Leucine Zipper (bZIP) family motifs; however, specific mechanisms of transcriptional regulation by these TFs to program TMEM are not fully understood. During TCR stimulation, transient upregulation of bZIP TF Batf3 is accompanied by binding of genomic loci encoding its motif. Nascent RNA sequencing of IL-2Rα-/- CD8+T cells shows that Batf3 upregulation upon TCR stimulation is highly dependent on IL-2R signaling. Constitutive loss of either IL-2Rα or Batf3 results in decreased survival of CD8+ T cells during the transition from effector to memory phase of acute LCMVArm infection, and reduced immunological memory towards infection re-challenge. We find that enforced overexpression of Batf3 cDNA using a retroviral vector in virus-specific IL-2Rα-/- CD8+ T cells greatly enhanced their capacity for survival into the contraction phase of primary infection. Upon secondary infection, ectopic Batf3 expression rescued the proliferation defect seen in IL-2Rα-/- CD8+ T cells. These results suggest that Batf3 plays a crucial role within the IL-2R signal transduction cascade during CD8+ T cell activation to promote enhanced cell survival into the memory phase of infection response.
Maternal hypertension alters offspring’s glial cell proportions and behavior
Authors: Johnson Morgan , Liu Yu , Brummond Daniel , Mikkelsen Emma , Bunton Olivia , Scroggins Douglas , Heck Detlef , Scroggins Sabrina
Published: November 2024

Preeclampsia (PreE), a hypertensive disorder during pregnancy, is a primary contributor to global obstetric morbidity and mortality. Its effects encompass heightened risks of cognitive impairment and behavior disorders in children born to mothers with PreE. The objective of our study was to determine whether PreE leads to early modifications in the brain's glial cell populations and/or behavioral changes in offspring. To induce PreE, C57BL/6J dams received vasopressin or saline through subcutaneous infusion throughout gestation. Following natural parturition, offspring were euthanized at various postnatal timepoints for glial cell analysis and behavioral evaluation. Flow cytometry was utilized to assess microglia and astrocyte populations. Offspring of PreE pregnancies exhibited decreased total microglia and astrocytes in the brain. Additionally, when compared to offspring from normotensive pregnancies, those born to mothers with PreE displayed impaired performance in the spontaneous alternation Plus-maze task, signifying altered spatial working memory. Our findings substantiate that PreE leads to diminished glial cell populations in offspring postnatally, and these mice exhibit changes in spatial working memory. Continuing research aims to elucidate the influence of PreE on the numbers and functioning of glial cells, along with examining how the compromised numbers and/or functionality of these cells affects the behavior and cognition of offspring born to mothers with PreE.
Evaluating the impact of influenza infection on pig lungs by single-cell RNA sequencing (scRNA-seq)
Authors: Madrid Darling Melany , Gu Weihong , May Alyssa , Touchard Laurie , Wen Yuhan , Driver John
Published: November 2024

Despite being the most widely consumed meat worldwide, the pig industry faces economic challenges from respiratory viruses including influenza. Pigs are also used as a reliable translational model for human respiratory disorders due to their similar respiratory tracts. However, limited pig-specific reagents have hindered understanding the pig’s immune system. Hence, here we used single cell RNA sequencing (scRNAseq) for unbiased transcriptional profiling of pig immune responses during influenza infection and oseltamivir antiviral therapy. Fourteen six-week-old mixed-breed pigs were intratracheally infected with pandemic 2009 H1N1 influenza virus. Seven of these pigs were treated for 5 days with oseltamivir. A control group was mock-infected. Lung samples were taken 5 days post-infection for scRNAseq, histopathology, and virus titers. We identified 24 pig leukocytes populations which were integrated with previously published human and mouse lung cells. Influenza virus infection/oseltamivir treatment altered the cellular composition, transcriptional networks, and cell-cell interactions in the lung. γδ T cells that are unique to pigs and abundant in the lung responded strongly to infection. Oseltamivir reduced antiviral and inflammatory responses. Our data provide a resource to better understand the pulmonary immune responses of pigs and advances using swine as a model for studying human pulmonary immune responses.
Simultaneous spatial profiling of mRNAs, cell surface proteins and cell surface protein protein interactions in human tonsil.
Authors: wang huili , Xia Junjie , Clark Marcus , Tay Savas
Published: November 2024

Protein-protein interactions are critical for the functionality of proteins and, consequently, for signaling events in numerous biological processes. High-throughput technologies capable of profiling these interactions at a spatial level are essential for deeper insights. In this study, we introduce Spatial Proximity-Sequencing (Sprox-seq), a novel technology that leverages proximity-sequencing with the 10x Visium platform for the concurrent detection of mRNAs, cell surface proteins, and protein-protein interactions. We utilized Sprox-seq to profile 20 proteins and their interactions along with whole transcriptomics in human tonsils. Our analysis revealed that protein-protein interaction-based clustering correlates well with RNA-based clustering and reflects the tissue's anatomical structure. Furthermore, we could distinguish the dark and light zones of germinal centers using differential protein expression and interaction patterns. Detailed examination of four distinct germinal centers revealed that protein and interaction data could identify one germinal center, which mRNA data could not achieve. Moreover, our study identified numerous differentially expressed protein interactions, including a range of previously uncharacterized interactions, indicating an important role in the development of germinal centers. Overall, our findings demonstrate that Sprox-seq can provide a multifaceted view of spatial molecular dynamics and discover new protein protein interactions.
Generating reliable custom MHC tetramers with the QuickSwitchTM tetramer platform.
Authors: Poluektov Yuri , Delcommenne Marc
Published: November 2024

Progress in immunology research depends on the continuous development of novel, more adapted and more user-friendly research tools. In 1996 Altman and Davis invented MHC class I tetramers for directly monitoring MHC/peptide specific CD8+ T cells by flow cytometry. Production of MHC tetramers is long and labor intensive and requires advance knowledge of the peptide sequences to be incorporated in these complexes. The recent Covid-19 pandemic has led to new vaccination strategies, which rely on selecting a collection of immunogenic peptides and generating mRNA constructs that contain these sequences. Peptide selection is primarily based on in silico MHC binding tools, which do not always give reliable results. After this first selection step it is necessary to validate MHC peptide binding. For this purpose, a dual use platform was devised, for both assessing MHC peptide binding in a semi-quantitative manner and for T cell staining. The assay is based on a placeholder peptide (or exiting peptide) that can be exchanged for higher affinity binding peptides. Kits with most frequent MHC class I or II alleles are available. Multiple MHC class I and II tetramers can quickly be generated and used for staining within a couple of days.
Development of a novel long-acting injectable, IVL4002 for modulation of multiple sclerosis: Pharmacokinetics and efficacy studies in an experimental autoimmune encephalomyelitis mouse model
Authors: Kim Ju Hee , Youm Soyoung , Lee Slgirim , Lee Mase
Published: November 2024

Multiple sclerosis (MS), a prevalent autoimmune disease and lacking a well-defined etiology and cures, drove the development of diverse disease-modifying therapies (DMTs). Despite the efficacy of DMTs and immunosuppressants, challenges persist, including adverse effects and varying response rates. A promising approach involves the utilization of a novel drug delivery system (DDS) for controlled drug release. Herein, we introduce a novel long-acting injectable (LAI) designed for MS modulation, IVL4002, developed using IVL-DrugFluidics™ manufacturing platform. IVL4002 comprises monodisperse microspheres encapsulating an FDA-approved drug which was discovered by drug repurposing. The selected drug has been utilized in the clinic for a non-MS CNS indication with a favorable safety profile and has also been used in autoimmune diseases off-label for decades. As shown in the preclinical pharmacokinetics studies, IVL4002 maintains drug levels within the target range over 4 weeks without a burst release. Combined with the advanced DDS, the drug was repurposed for MS management, of which the efficacy was shown in experimental autoimmune encephalomyelitis mice by reducing the pro-inflammatory cytokine release. In conclusion, our physicochemical and pharmacological studies underscored the potential of IVL4002 to be used as a 1-month LAI for MS management. Positive results have led to the approval of a Phase 1 clinical trial and it is expected to begin in the second-half of 2024.
TLR7-agonist nanoparticle adjuvant modulates germinal center responses and promotes broad antibody responses against heterologous viral strains of influenza and SARS-CoV-2
Authors: Yin Qian , Luo Wei , Pulendran Bali , Davis Mark
Published: November 2024

The ideal vaccine against viruses such as influenza and SARS-CoV-2 must provide robust, durable, and broad immune protection against multiple viral variants. However, antibody responses to current vaccines often lack robust cross-reactivity. To tackle this challenge, we develop a polymeric Toll-like receptor 7 agonist nanoparticle (TLR7-NP) adjuvant, which enhances lymph node targeting, promotes persistent immune cell activation, and leads to early germinal center responses. When mixed with alum-adsorbed antigens, this TLR7-NP adjuvant elicits cross-reactive antibodies for both dominant and subdominant epitopes in mice. This TLR7-NP-adjuvanted influenza subunit vaccine successfully protects mice against the viral challenge of a different strain. This strategy also enhances the antibody response to a SARS-CoV-2 subunit vaccine against multiple viral variants that have emerged. Moreover, this TLR7-NP augments antigen-specific immune responses in human tonsil organoids. Overall, we describe a nanoparticle adjuvant to improve immune responses to viral antigens, with promising implications for developing broadly protective vaccines.
Antibody profiling of COVID-19 patients reveals antibodies and autoantibodies that may be associated with faster recovery from COVID-19
Authors: Onabajo Olusegun , Budde Petra , Kuebler Peter , Bräutigam Manuel , Haridha Shivram , Stubbings William , Tsai Larry , Zucht Hans-Dieter , Chandler G Scott , McBride Jacqueline , Bauer Rebecca
Published: November 2024

COVID-19 infection exhibits a broad spectrum of clinical outcomes and a combination of host and viral factors interact to drive variability in COVID-19 outcomes. While some studies showed severe COVID-19 patients were more likely to have neutralizing autoantibodies to type-I IFNs relative to the general population, other studies showed preexisting immunity to endemic coronaviruses could be beneficial. This suggests comprehensive evaluation of antibodies and autoantibodies associated with COVID-19 outcomes could reveal important host factors contributing to disease outcomes. Here we profiled antibodies to 87 bacterial and viral proteins, including SARS-CoV-2, and autoantibodies to 383 immune- and autoimmune-associated antigens using the Oncimmune serological profiling technology. A total of 460 patients from two COVID-19 clinical trials (COVACTA, NCT04320615, and MARIPOSA, NCT04363736) were used in this study, and baseline levels of antibodies and autoantibodies in serum samples were associated with COVID-19 outcomes. We observed higher autoantibodies to type-I IFNs and some chemokines were associated with longer time to hospital discharge (TTHD), while higher autoantibodies to IL-6 and IL-36A led to faster TTHD. High levels of antibodies to endemic coronaviruses and seasonal influenza were also associated with faster TTHD. These findings suggest preexisting host antibody and autoantibody repertoire could provide some protection from the more severe consequences of COVID-19.
Fate mapping LAG3+ exhausted CD8+ T cells.
Authors: Aggarwal Vaishali , Liu Chang , Jian Cui , Wang Haiguang , Manne Sasikanth , Vignali Kate M , Wherry Edward , Workman Creg , Vignali Dario
Published: November 2024

T cell exhaustion, a state of T cell dysfunctionality is well defined in chronic viral infections, however, not completely understood in tumor setting. The exhausted T cells (TEX) express high levels of LAG3, an inhibitory receptor regulating T cell exhaustion. We hypothesize defining lineage heterogeneity and plasticity of LAG3+ T cells, would be insightful in determining fate and functionality of these cells. We generated a novel lineage tracing Lag3iCreERT2Rosa26LSL-tdTomato mouse strain facilitating temporal assessment of LAG3+ cells using tamoxifen induced pulse/chase scheme. This enables fate-mapping of ‘bona fide’ tumor-reactive LAG3+ cells and understanding their transcriptional and epigenetic profiles. We identified two distinct LAG3- tdT+ and LAG3+ tdT+ exhausted T cell populations characterized with tdT+ expression. These two distinct tdT+ subsets are transcriptionally, and epigenetically distinct. The LAG3- tdT+ cells are derived from cleavage of surfaceLAG3 and are ‘stem-like’ exhausted CD8+ T cells with intermediate PD-1 expression. These ‘stem-like’ exhausted CD8+ T cells persist in vivo, have differentiation plasticity and contribute to peripheral tumor-specific memory. The other subset, LAG3+ tdT+ T cells are ‘terminally exhausted’, restricted to tumor microenvironment and express high levels of PD-1, TIM-3 and TIGIT. Overall, this model provides a unique advantage of characterizing LAG3- tdT+ ‘stem-like’ exhausted CD8+ T cells.
Twenty-Color Immuno-Analysis of Tumor-Infiltrating Lymphocytes in Lung Cancer
Authors: Ma Weide , Ma Yanhong , Yue Danqi , Anderson Keon , Chu Dongdong , Zhang Yaxuan , Zhang Long , Deng Jun , Edinger Mark , Wang Xiaoyang (Alice)
Published: November 2024

Tumor-infiltrating lymphocytes (TILs) are naturally occurring immune cells residing in the solid tumor microenvironment. Some of these TILs are tumor specific and may possess cytotoxic anti-tumor activity. The harvesting, selection, expansion, and reinfusion of these cells represent a current therapeutic approach that may prove to be safe and effective.

We developed an immunophenotyping panel to study the tumor microenvironment in human lung cancer specimens. This is a broad panel that includes blood and epithelial cell markers. Twenty lung cancer samples were collected following institutional protocol and dissociated into single cells within 24 hours of resection. Cells were then stained and data was acquired on a 3 laser Cytek™ Aurora™ spectral flow cytometer.

The frequencies of cell populations were measured. Increased percentages of intermediate and non-classical monocyte subsets were observed. PD-L1 was detected on neoplastic epithelial cells and EMT (epithelial-mesenchymal transition) cells. Interestingly, a population of CD45 and EpCAM double positive cells was detected in some samples. These cells expressed both epithelial and macrophage markers, suggesting a macrophage lineage origin with abnormal expression.

This streamlined single cell sample preparation and multiparametric panel provide an efficient method for the analysis of the tumor microenvironment. Expansion of the sorted cytotoxic TILs could potentially be reinfused into a patient for cancer treatment.
Silicosis and silica-induced autoimmunity in multiple Collaborative Cross strains.
Authors: de Ocampo Caroline , Mayeux Jessica , Kono Dwight , Pollard Michael , Janssen Lisa
Published: November 2024

Background: The connection between pulmonary silica dust exposure and the development of systemic autoimmunity is well established. However, the extent to which genetic differences influence the severity of silica-induced autoimmunity remains unclear.

Objective: Investigate the role of silica exposure on disease biomarkers (cytokines/chemokines and antinuclear antibodies (ANA)) and systemic autoimmune disease in genetically diverse Collaborative Cross recombinant inbred (CC RI) mice.

Methods: Baseline serum ANA and chemokines/cytokines were assessed from 61 CC RI strains. Six CC RI strains, chosen to represent different baseline immune phenotypes, were exposed to 5 mg crystalline silica or PBS and examined 12 weeks later for serum ANA, bronchoalveolar lavage fluid cell counts, tracheobronchial lymph node and spleen weights, and histological assessment of silicosis and lupus nephritis.

Results: All strains developed silicosis; however, two strains with low baseline immune activity did not develop silica-induced autoimmunity. In contrast, strains with higher baseline immune activity developed elevated ANA levels with strain variation in ANA types. One strain developed lupus nephritis, with significantly greater severity compared to controls.

Conclusion: Baseline immune activity significantly influences the development of silica-induced autoimmunity and lung inflammation; however, genetics may be mediating silicosis and systemic autoimmunity independently.
Arid5a-dependent RNA translation underlies pathology in IL-17-driven autoantibody-induced glomerulonephritis
Authors: Li Yang , Vyas Shachi , Mehta Isha , Asada Nariaki , Dey Ipsita , Taylor Tiffany , Bechara Rami , Amatya Nilesh , Aggor Felix , Coleman Bianca , Li De-Dong , Yamamoto Kenta , Ezenwa Ogechukwu , Sun Yeque , Sterneck Esta , McManus C Joel , Panzer Ulf
Published: November 2024

Autoantibody-mediated glomerulonephritis (AGN) results from kidney-damaging autoimmune responses, and there is urgent need for better treatment. Emerging studies implicate Th17 cells in AGN. IL-17 signals in part through the noncanonical RNA binding protein Arid5a, which we show is induced in renal epithelial cells (RTECs) in an IL-17-dependent manner during experimental AGN. Arid5a is undetectable in healthy kidney but elevated in human glomerulonephritis biopsies. Arid5a-/- mice were refractory to AGN. Mice lacking C/EBPd but not IkBz were protected from AGN. Although Arid5a bound to Cebpd mRNA, Arid5a-deficiency did not alter cellular Cebpd mRNA concentrations, whereas C/EBPd protein levels were substantially suppressed. In line with this, global protein synthesis was suppressed in RTECs lacking Arid5a. Transcriptome-wide RIP-Seq analysis in RTECs and stromal cells showed that IL-17 prompts Arid5a to bind to inflammatory mRNAs. Additionally and unexpectedly, Arid5a interacted with ribosomal RNAs (rRNAs) and other transcripts involved in protein translation. After IL-17 treatment, Arid5a was enriched in the cytoplasm and associated with the ribosome through interactions with 40S and 60S rRNAs. Accordingly, Arid5a drives IL-17-induced renal autoimmunity through post-transcriptional control of RNA at the level of translation and ribosome interactions, providing mechanistic insight into how cytokines mediate inflammation.
TGFb-JMJD3 pathway regulates fibroblast to myofibroblast transition in wound repair
Authors: Bauer Tyler , Mangum Kevin , Barrett Emily , Shadiow James , Joshi Amrita , Buckley Samuel , Wasilowski Rachel , Moore Bethany , Gudjonnson Johann , Davis Frank , Gallagher Katherine
Published: November 2024

In normal wound healing, resident fibroblasts transition to myofibroblasts, which function to contract and close wounds. In pathologic conditions where wounds fail to heal (such as diabetes), fibroblasts do not increase myofibroblast gene expression; this mechanism is poorly understood. Using human wound scSeq and murine transgenic models, we examined the epigenetic mechanisms that may regulate this transition in the setting of injury. First, we identify that the cytokine TGFb increases myofibroblast genes in vitro. We performed an epigenetic superarray on TGFb-stimulated dermal fibroblasts, where JMJD3, an H3K27 demethylase, was significantly upregulated and was also confirmed by qPCR. ScSeq of human wounds identified JMJD3 as strongly upregulated in myofibroblasts. ChIP identified decreased H3K27me3, the activating mark of JMJD3 at myofibroblast genes further confirming its role in regulating myofibroblast genes. SiRNA knockdown of JMJD3 led to reduced myofibroblast gene expression. These findings were confirmed in a novel, tamoxifen induced, fibroblast specific knockout of JMJD3 unique to our lab. An unbaised superarray focused on remodelling and cell adhesion pathways identified that these mice have deficient levels of many structural and remodelling genes. Murine wounds were found to upregulate JMJD3 compared to distant skin, which was less pronounced in diabetic wounds. This work identifies JMJD3 as a TGFb-mediated regulator of fibroblast to myofibroblast transition.
Lanthionine synthetase C-like receptors reduce eosinophilia, type 2 immune responses and lung inflammatory pathology in mouse models of asthma
Authors: Leber Andrew , Hontecillas Raquel , Tubau-Juni Nuria , Bassaganya-Riera Josep
Published: November 2024

Airway hyperresponsiveness and asthma are associated with altered glucose metabolism with inhibition of anaerobic glycolysis reducing inflammation and lung eosinophilia. LANCL receptors are key focal points for metabolism in immune cells, controlling mitochondrial mass and oxidative pathways all while promoting immunoregulatory differentiation. Knockout of LANCL receptors leads to diminished Treg differentiation, altered epithelial cell metabolism and impaired phagocytic clearance, all of which can contribute to the chronicity of asthma. We developed an oral agonist for LANCL receptors and tested it within ovalbumin and house dust mite induced models of asthma in mice. Treatment in vitro with the agonist resulted in reduced differentiation of inflammatory and allergic CD4+ T cells and reduced production of inflammatory cytokines in macrophages. In the OVA model, oral treatment with an LANCL therapeutic, beginning on the second day of aerosol challenge significantly reduced neutrophilia and eosinophilia in the lung by flow cytometry and histology. LANCL treatment significantly reduced anti-OVA IgE titers and type 2 cytokines, IL-5 and IL-13. Reduction of eosinophilia and type 2 immune responses were validated in the house dust mite model. In both models, lung gene expression trends were consistent with improved pulmonary function and mitochondrial metabolism. Thus, activation of LANCL receptors is a feasible strategy for the treatment of asthma and pulmonary inflammation.
Amplified STAT3 increases expression of the CD39 ectoenzyme and impairs function of CD8+ T cells.
Authors: Campos Duran Jose , Sayed Samir , Knight Montana , Dalalo Megan , Conrey Peyton , Hay Ceire , Schmitt Erica , Thelin Martin , Mauracher Andrea , Forbes Lisa , Holland Steven , Bergerson Jenna , Su Helen , Romberg Neil , Freeman Alexandra , Leiding Jennifer , Anderson Mark
Published: November 2024

Regulation of T cell signaling is paramount to mount an appropriate immune response and protect the host from pathophysiology. Here, we study the impact on CD8+ T cells in monogenic inborn error of immunity patients exhibiting dysregulated signal 3 (cytokine signaling) pathways resulting from STAT3 gain- or loss-of-function variants (STAT3 GOF and STAT3 LOF). Using high dimensional immune profiling, we demonstrate increased expression of the ectoenzyme, CD39, which hydrolyzes extracellular ATP, on STAT3 GOF patient CD8+ T cells. Moreover, CD8+ T cells from two mouse models of STAT3 GOF (STAT3+/G421R and NOD-STAT3+/K392R) recapitulated this increase in CD39. In vitro, STAT3-activating cytokines (e.g., IL-6, IL-21, IL-27, IL-10) augment CD39 induction in a TCR-dependent manner in healthy donor and in STAT3 GOF CD8+ T cells. In contrast, STAT3 LOF patients show diminished CD39 induction. We find a positive correlation between CD39 induction and phosphorylated STAT3 levels while STAT3 inhibition prevents this cytokine-driven augmentation of CD39. Finally, acutely activated, and sorted CD39+CD8+ T cells from healthy donors inhibit bystander CD8+ T cell cytokine production (e.g., IFN-g). Pharmacologic inhibition of CD39 enzymatic activity or genetic ablation via CRISPR/Cas9 editing reversed decreases in cytokine production. Together, our data demonstrate a mechanistic connection between amplified STAT3 signaling and CD39 levels which is capable of impairing CD8+ T cell function.
Spatial Molecular Profiling of the Kidney of Children with Lupus Nephritis
Authors: Rodriguez-Alcazar Juan , Balaji Uthra , Troncoso Leanna , Reid Amanda , Hamon Pauline , Houghton Sean , Walters Lynnette , Smitherman Cynthia , Baisch Jeanine , Santos Marina , Larosiliere Owen , Robinson Lauren , Wright Tracey , Merad Miriam , Caielli Simone , Gu Jinghua , Pascual Virginia
Published: November 2024

Childhood-onset lupus nephritis (cLN) affects 80% of pediatric systemic lupus erythematosus patients. Classified based on histological renal features, cLN is treated with immunosuppressive agents that trigger side effects. Despite the need for targeted therapies, little is known on the molecular pathways causing cLN and their variability across patients. We employ spatial transcriptomics and proteomics approaches to profile kidney biopsies of cLN patients and define cellular and molecular endotypes across clinical traits. We found that distinct spatial organization of the renal immune compartment classified cLN biopsies according to histological classification. Myeloid cells with phagocytic and highly inflammatory transcriptional programs, including the expression of S100A9, infiltrate glomeruli of proliferative cLN. A subset of these patients also presents glomerular inflammasome-competent myeloid cells expressing IL-1β. Tubulointerstitial and periglomerular immune hubs characterize a subgroup of membranous and mixed cLN. Immune hubs’ cell composition varies across patients and tissue lesions and can be skewed towards specific lymphoid populations, including T, B, or plasma cells with different isotype specificity. Our results highlight the importance of understanding the spatial organization and heterogeneity of the renal immune compartment in cLN. The prognostic value of these findings and their relevance to target disease is being currently explored.
Cell-intrinsic Tim-3 is Required for an Optimal Acute CD8+ T Cell Response
Authors: Manandhar Priyanka , Landy Emily , Cardona Eddie , Kane Lawrence
Published: November 2024

Tim-3, or transmembrane immunoglobulin and mucin domain-3, is a type I membrane protein expressed by various immune cells, best known as a negative regulator of anti-tumor immunity. However, Tim-3 also has a co-stimulatory role in T cells under some conditions, via enhancement of PI3K signaling. We hypothesize that Tim-3 signaling enhances CD8+ T cell activation during acute infection and contributes to enhanced CD8+ effector function.

To test this hypothesis, we used LCMV-specific TCR transgenic (P14) mice expressing a truncated, non-signaling, allele of Tim-3 or CD8-specific deletion of Tim-3 and used LCMV Armstrong as an acute infection model. At the effector stage, endogenous Tim-3+ T cells had increased expression of effector markers, and cytokine production compared with Tim-3- cells. Tim-3 knockout mice also showed a significantly lower number of LCMV-specific T cells. Bulk RNA sequencing and TCR sequencing revealed that Tim-3-expressing effector CD8+ T cells had a distinct pattern of effector gene upregulation, with increased expansion of a subset of TCR clones. Mechanistically, Tim-3 signals enhances phosphorylation of Foxo1, inhibiting its entry into the nucleus, conferring enhanced effector function. Thus, Tim-3 signaling contributes positively to an effective CD8+ T cell response during acute infection. These findings may lead to a better understanding of CD8+ T cell function in different settings, including in tumors.
Investigating neuroinflammation in the human brain tumor microenvironment using the new RNAscopeTM Multiomic assay
Authors: Wakhloo Debia , Dikshit Anushka , Kim Ge-Ah , Yu Julia , Deshpande Sonali , Wang Li-Chong , Srinivasan Maithreyan
Published: November 2024

Glioblastomas are one of the most aggressive forms of brain tumors characterized by distinct genetic and molecular signatures. Compared to other solid tumors, role of immune cells in progression, invasion and prognosis is not well studies for CNS tumors. Here, we demonstrate a novel RNAscope Multiomic Assay that enables detection of multiple RNA and protein markers simultaneously.

With this novel TSA amplification-based co-detection assay we visualized a few combinations of 3 RNA and 3 protein marker panels on human FFPE normal brain and brain tumor tissues. Antibodies targeting key immune cell markers and morphology markers such as CD8, IBA1 ,CD68, S100B and GFAP were used. RNA probes targeting chemokines and cytokines such as CXCL10, IFNG, TNFA, CXCL2. IL-6 were also used in the panels.

Immune cells infiltrating the brain tumor tissues were characterized by studying co-expression of key RNA and protein markers. The tumors demonstrated infiltration of immune cells such as T cells, microglia and macrophages. Expression of key cytokines indicated that some populations within these cells were activated. Distinct differences in neuroinflammation signatures were also observed between the normal and tumor brain tissues.

The multiomic assay offers a powerful technique for visualizing target RNA biomarkers in specific cell-types identified by cell-marker protein expression.
Synthetic saponin derivatives act as functional subunit vaccine adjuvants through an NLRP3-inflammasome-independent mechanism
Authors: Santhin Austin , Ghorai Jayanta , Hartwig Stacey , Miller Ann , Almounajed Leila , Nguyen Dr Hien , Varga Dr Steven
Published: November 2024

Quillaja saponaria 21 (QS-21) is a saponin compound extracted from the bark of a South American evergreen tree. The ability of QS-21 to induce robust cellular and humoral immunity as an adjuvant led to it being evaluated in multiple clinical vaccine trials. However, mechanistic and toxicity questions following its purification hinder its potential use and led to the search for suitable alternative adjuvants. A group of 14 novel synthetic compounds have been chemically derived from a less toxic saponin, lablaboside F, to be tested as suitable alternatives to QS-21. When administered with an OVA peptide antigen intramuscularly into C57BL/6 mice using a prime-boost strategy, compound JG-151 elicited increased production of OVA-specific total IgG antibodies 2 weeks post-boost compared to control mice. When analyzed in vitro, JG-151 displayed decreased cellular toxicity 24 hrs post-treatment compared to QS-21. However, JG-151 neither activated NF-kB alone nor induced significant production of the pro-inflammatory cytokine IL-1ß with MPLA 24 hrs post-treatment. These results indicate JG-151 does not activate the NLRP3 inflammasome, in contrast to QS-21. Our results demonstrate that novel saponin derivatives, such as JG-151, safely elicit humoral immunity through an NLRP3 inflammasome-independent mechanism and could serve as suitable subunit vaccine adjuvant alternatives to QS-21.