bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial Analysis of NETotic Neutrophil Crosstalk with Monocytes/Mφ in Oral Squamous Cell Carcinoma
Authors: Lowery Austin , Zhao Juanjuan , Chiang Austin , Zhu Yanfang
Published: November 2024

Neutrophils are specialized immune cells that exhibit dual roles in cancer immunity; both pro and anti-tumor functions have been characterized thus far. It is now recognized that previously identified Myeloid-Derived Suppressor Cells (MDSCs) that impede anti-tumor immunity in various cancers are comprised of pathologic monocyte and neutrophil types. However, the interplay between tumor-associated neutrophils, monocytes, and their impact on tumor immunity remains unclear. Neutrophils execute their immune functions via NETosis, an exceptional mechanism that extrudes neutrophil extracellular traps (NETs), which play a dual role in cancer. In some cancers, NETs recruit macrophages toward an anti-tumor response, yet prolonged interaction induces apoptosis, contributing to immune suppression in the tumor microenvironment (TME). In oral squamous cell carcinoma (OSCC), linked to poor prognosis and high mortality, NETotic neutrophil infiltration at the tumor site correlates with improved survival. Conversely, heightened neutrophil presence in late-stage metastatic lymph nodes reduces survival. Consistently, elevated NET concentration in patient serum was identified. In this study, we use CosMx Spatial Molecular Imager (CosMx SMI) to investigate in-depth transcriptome changes of monocytes/Mφs induced by crosstalk with NETotic neutrophils in the tumor site from MOC2 tumors (OSCC mouse model) to demonstrate the contribution of NETotic neutrophil-monocyte/Mφ crosstalk to OSCC initiation.
Role of lipid droplets and prostaglandinE2 in Coxiella burnetii intracellular growth.
Authors: Biancaniello Maria , Mulye Minal
Published: November 2024

The obligate intracellular bacterium, Coxiella burnetii, causes human Q fever. Infection with Coxiella via inhalation presents as atypical pneumonia which may relapse years later, leading to potentially fatal endocarditis. Our goal is to determine the mechanisms Coxiella employs for long-term survival in the host. While the bacterium initially infects alveolar macrophages, in endocarditis patients Coxiella is found in foamy macrophages rich in neutral lipid storage organelles, lipid droplets (LDs). Our studies show that blocking LD breakdown inhibits bacterial growth suggesting that LD-derived lipids are crucial for Coxiella’s survival. LD breakdown releases arachidonic acids, precursors for the lipid immune mediator prostaglandinE2 (PGE2) which promotes immunosuppression in alveolar macrophages. We hypothesize that Coxiella manipulates host cell LD metabolism to promote a PGE2-mediated immunosuppressive environment and survive long-term in the host. To test this, we quantified PGE2 production in Coxiella-infected cells with and without LD breakdown inhibitor, atglistatin. ELISA showed a LD breakdown-dependent increase in PGE2 levels. Fluorescence microscopy revealed PGE2 production blockade with COX-2 inhibitors significantly decreased bacterial growth which was rescued by exogenous PGE2 suggesting that PGE2 promotes Coxiella survival. Ongoing studies are identifying the correlation between LDs and PGE2 production and the role of LDs in immunosuppression.
Intestinal helminth infection impairs vaccine-induced T cell responses and protection against a SARS-CoV-2 Omicron variant in mice
Authors: Desai Pritesh , Karl Courtney , Ying Baoling , Liang Chieh-Yu , Garcia-Salum Tamara , Santana Ana Carolina , Urban Jr Joseph , Elbashir Sayda , Edwards Darin , Thackray Larissa , Sekaly Rafick , Diamond Michael
Published: November 2024

Although vaccines have reduced the global COVID-19 disease burden, the impact of gastrointestinal helminth infection on their efficacy in endemic regions of the world remains uncertain. Here, using Heligmosomoides polygyrus bakeri (Hpb), a model, non-invasive murine intestinal hookworm, we evaluated whether Hpb infection affects the efficacy of mRNA S6P, a preclinical vaccine encoding a hexaproline stabilized spike of the Wuhan-1 strain of SARS-CoV-2. In the setting of Hpb infection, immunization of mRNA S6P generated equivalent serum antibody and antigen-specific B cell responses. However, antigen-specific CD4+ and CD8+ T cell responses were reduced compared to mice immunized without Hpb infection. Helminth infection did not skew vaccine-induced T cell responses but rather inhibited their capacity to produce polyfunctional cytokines. Hpb infection caused similar T cell defects in mice immunized with an approved adenoviral vectored SARS-CoV-2 vaccine (Ad26.COV2.S). Challenge experiments showed that although Hpb-infected mice vaccinated with mRNA S6P were fully protected against the ancestral SARS-CoV-2 strain WA1/2020, there was a loss of vaccine-mediated protection against Omicron variant BA5.5 compared to animals immunized in the absence of Hpb infection. These results in mice suggest that intestinal helminth infection can compromise T cell responses and protective efficacy of vaccines against SARS-CoV-2 variants that escape neutralizing antibody responses.
Exploring engineered HLA-I chaperones for enhancing the immunogenicity of cold tumors.
Authors: Hwang Daniel , Sun Shirley , Sgourakis Nikolaos
Published: November 2024

Immune surveillance relies upon efficient production of peptide loaded HLA class I molecules (pHLA-I), a process mediated by the antigen processing and presentation (APP) pathway. Accordingly, dysregulation of the APP pathway is a common immune evasion mechanism in cancers. This is especially true for “cold tumors”, a class of cancers with low immunogenicity, typically caused by global downregulation of pHLA-I and/or low mutational burden, resulting in immune escape and overall worse prognosis. We aimed to develop a method for restoring immunological recognition of this class of tumors by leveraging endogenous mediators of the APP pathway. We modified two important HLA-I chaperones, tapasin and TAPBPR, which have essential functions in stabilizing HLA-I molecules and in refining the repertoire of presented antigens. We have engineered variants of these chaperones, including HLA allotype-agnostic variants, which travel along the secretory pathway together with HLA molecules, enabling them to exert their chaperoning function in non-native subcellular compartments, including at the cell surface. We show that these Chaperone Fusion Proteins (CFPs) can increase HLA-A2 expression in multiple cell lines, including in a bona fide cold tumor cell line. Moreover, CFP expression resulted in enhanced T cell recognition and killing of target tumor cells. These data suggest that CFPs may be developed into a platform for enhancing the immunogenicity of cold tumors.
Modulation of antigen presentation by first-in-class ERAP1 and ERAP2 inhibitors for the treatment of cancer and autoimmunity
Authors: Clark Kristopher , Anderton Kate , Paes Wayne , Green Daniel , Khan Faraz , Ternette Nicola , Lam Tsang Wai , Wat Jesse , Bras Maaike , Trelber Tobias , Pfeiffer Anamarija , Redfern Dan , Rozo Annaig , Sabouni Joud , Garside Samantha , Kay Steven , Shiers Jason
Published: November 2024

The aminopeptidases ERAP1 and ERAP2 play a central role in the processing of antigens for loading onto MHC-I complexes with links to both autoimmunity and cancer. On one hand, genetic variants of each enzyme are associated with the development of several autoimmune disorders with the formation of autoantigens by ERAP1 and ERAP2 as a leading hypothesis for the mechanism behind this association. On the other hand, it is well established that patients with tumors characterized by high antigen diversity have increased response rates to immune checkpoint inhibitors. It follows that modulation of the cancer immunopeptidome offers a new therapeutic strategy to generate de novo anti-tumor T-cell responses and overcome current challenges of existing immuno-oncology (IO) therapeutics. Collectively, this knowledge implies that ERAP1 and ERAP2 represent a novel category of therapeutic targets for autoimmunity and immuno-oncology that can be manipulated to drive changes in antigen repertoire and thereby modulate the detection of cancer cells and self. Here, we present the characterization of our potent and selective First-in-Class ERAP1 and ERAP2 inhibitors both in vitro and in vivo. Importantly, we apply a state-of-the-art immunopeptidomics pipeline to define the qualitative and quantitative effects of these inhibitors. Our data demonstrate that ERAP1 and ERAP2 have overlapping and distinct effects on the antigen repertoire, consistent with their respective substrate specificities.
Modulation of IL-13 signal in allergic asthma.
Authors: Shankar Archana , McAlees Jaclyn , Hargis Julie , Lewkowich Ian
Published: November 2024

Allergic asthma, a chronic airway inflammatory disease affecting over 300 million people worldwide, is primarily driven by Th2 cytokines such as interleukin-13 (IL-13). Although IL-13 signals through a Jak1/Tyk2/STAT6-driven pathway, activation of other pathways has been reported (e.g., STAT3, SRC). To better understand the totality of signaling intermediates activated by IL-13, we employed an unbiased, phospho-proteomics approach. To this end A549 cells, a human airway epithelial cell line, were stimulated with IL-13 for 15 minutes, and peptides containing a phosphorylated Serine, or Threonine reside were identified by mass spectrometry. >1,200 individual phospho-peptides were identified, and 108 residues were found to be differentially phosphorylated. Based on string DB results, SRC (a non-receptor tyrosine kinase) was identified as a potentially novel IL-13-induced signaling intermediate activated in response to IL-13. Kinetic assessment of SRC activation suggested that it is activated within seconds of IL-13 stimulation, and that its activation preceded activation of canonical signaling intermediates like STAT6. Functionally, SRC antagonists reduced expression of multiple IL-13-induced gene in IL-13-treated A549 cells. Collectively, these data suggest that SRC may represent a novel signaling intermediate activated in response to IL-13, and that SRC antagonism may have therapeutic benefit in the treatment of asthma.
Caveolin-1 restrains pathogenic T follicular helper cell response in primary Sjögren’s syndrome
Authors: Lin Xiang
Published: November 2024

T follicular helper (Tfh) cells play a central role in humoral autoimmunity, including primary Sjögren’s syndrome (pSS). However, targeting Tfh cells is challenging in clinical management. Previous studies suggested that inducible T­cell co­stimulator (ICOS) directed Tfh cell motility in engaging bystander B cells, which promoted plasma cell differentiation and antibodies production. Here, we identified a novel function of caveolin-1 (Cav-1) in restraining ICOS expression in Tfh cells and pSS pathogenesis. Cav-1 deficiency significantly promoted human and murine Tfh cell responses, while Cav-1-/- mice exhibited exacerbated disease pathology of experimental SS (ESS). Peroxisome proliferator-activated receptor alpha (PPARa), a downstream transcription factor of Cav-1, rapidly repressed Icos transcription upon Tfh polarization, interestingly, independence of lipid metabolism. Phenotypic analyses suggested that Cav-1 and PPARa expressions were decreased in CD4+ T cells from pSS patients and ESS mice. Notably, pharmaceutical activation of PPARa with fenofibrate could suppress human and murine Tfh cells both in vitro and in vivo, while oral administration of fenofibrate effectively ameliorated ESS pathology in mice with acute or chronic inflammation. These results revealed an unrecognized role of Cav-1/PPARa axis in Tfh cell tolerance and pSS pathogenesis, suggesting PPARa as a promising target in the treatment of humoral autoimmunity.
Immunization with Sez6L2 protein mimics the human condition of late-onset cerebellar ataxia with associated Sez6L2 autoantibodies in mice
Authors: Reyes-Sepulveda Carlos , Randolph John , Hobbins Alison , Granato Julia , Ryan Allison , Silver Nina , Stout Angela , Hammond Jennetta
Published: November 2024

Sez6L2 autoantibodies have been identified in a subset of human patients with progressive cerebellar ataxia. Sez6L2 is a transmembrane protein expressed by most neurons, including Purkinje cells and granular cells of the cerebellum. Patients with Sez6L2 autoantibodies present with gait ataxia, slurred speech, and ocular motor symptoms. Some patients also develop limb ataxia and cognitive deficits. These case studies have led to the hypothesis that autoimmunity against Sez6L2 can directly cause cerebellar damage leading to ataxia. We tested whether C57BL/6J mice immunized against Sez6L2 protein develop ataxia symptoms. We also investigated the systemic immune response against Sez6L2 and whether there was increased immune cell infiltration into the brain. We found that mice immunized with Sez6L2 generated significant levels of anti-Sez6L2 antibodies, in addition to a significant population of Sez6L2-specific CD4 and CD8 T cells. Furthermore, these mice had increased CD4+ T cell infiltration into the brain. The Sez6L2 immunized mice also exhibited mild mobility impairments in open field and wire grid walk assays. Overall, our results demonstrate that an autoimmune response to Sez6L2 in mice can lead to mobility impairments and pathology consistent with human patients who develop cerebellar ataxia associated with Sez6L2 autoantibodies. This new mouse model should be useful for mechanistic studies on a poorly understood autoimmune disease.
The impact of pediatric obesity on T cell function
Authors: Hay Ceire , Sayed Samir , Knight Montana , Campos Jose , Nelson Molly , Abrams Eric , Gunnala Pooja , Gonzalez Elena , Espinoza Diego , Beers Jarad , Collins Joy , Ruffner Melanie , Henao-Mejia Jorge , Vahedi Golnaz , Dumon Kristoffel , Prout Elizabeth , Henrickson Sarah
Published: November 2024

The increasing prevalence of childhood obesity is an urgent public health crisis. Children with obesity (≥ 95th percentile BMI for age and sex) are at an increased risk for developing asthma as well as cardiometabolic and autoimmune disorders. However, the mechanisms by which excess weight impacts immune function in children is incompletely understood. To address this gap in knowledge, we assessed changes in immune cell function in obese children before and after bariatric surgery, a procedure used to facilitate weight loss in patients with severe obesity. We integrated high dimensional spectral flow cytometry, in vitro functional assays, and single-cell RNA-sequencing of intra-operative visceral adipose tissue and PBMCs to find that CD8+ T cells from obese children, compared to age-matched controls, had increased expression of TOX and inhibitory receptors (PD-1, LAG-3). This dysfunctional phenotype was persistent 6-18 months after surgery, despite significant weight loss (average change in BMI of 17%). Moreover, baseline CD8+ T cells from obese patients exhibit impaired proliferation and altered metabolic profiles following TCR stimulation. Future work will determine functional differences in the peripheral T cells of bariatric surgery patients at baseline compared to 6-18 months post-surgery and assess the local immune landscape within visceral adipose depots from obese compared to non-obese children.
Soluble Plasma Proteins of Tumor Necrosis Factor and Immunoglobulin Superfamilies Reveal New Insights into Immune Regulation in People with HIV and Opioid Use Disorde
Authors: Ghanta Priya , Dang Christine , Nelson Mindy , Feaster Daniel , Forrest David , Pahwa Rajemdra , Tookes Hansel , Pallikkuth Suresh , Pahwa Savita
Published: November 2024

Opioid Use Disorder (OUD) is present in high frequency in people with HIV (PWH). This study investigated proteins of the TNF and Immunoglobulin superfamilies (SF) in relation to Flu vaccine responses in PWH with and without OUD. 210 participants (median age 46 years) were recruited into OUD groups: HIV+OP+ and HIV-OP+, and non-opioid users: HIV+OP- and HIV-OP-. Flu vaccine titers post-vaccination at 21-28 days (T2) and fold change (T2/T0) were higher in people with OUD compared to PWH without Opioid use, while being equivalent to controls (HIV-OP-). 48 soluble molecules of the Tumor necrosing factor (TNF) and Immunoglobulin (Ig) superfamilies (SF) were analyzed in plasma by multiplex Luminex technology. Both OP+ groups (HIV+OP+ and HIV- OP+) had higher co-stimulatory TNF SF molecules including 4-1BB, OX-40, CD40 that were positively correlated with Flu vaccine responses. The HIV+ OP- group displayed a different profile, with high Ig SF molecules including PD-L2, CTLA-4 and Perforin, of which PD-L2 was negatively associated with Flu vaccine titers. The higher Flu vaccine response in the groups with OUD (HIV+OP+ and HIV- OP+) could be attributed to high levels of specific plasma proteins of the TNF superfamily that are known to be important for T cell activation, proliferation, and T-B interactions needed for antibody responses and could have negated the immunosuppressive effects of HIV. These findings could lead to development of new diagnostic and therapeutic tools.
Interferon responses in monocytes at the febrile phase of PFAPA
Authors: Seo Euri , Kim Youngun , Koh June Young , Kang Ji-Man , Shin Eui-cheol
Published: November 2024

Periodic fever, aphthous stomatitis, pharyngitis, and adenitis, generally called PFAPA, is a disease in which symptoms like the name of the disease appear with a regular periodicity. Each fever event can be ended with one dose of steroids. To identify its pathogenesis, which remained unknown, we performed single-cell mRNA sequencing using PBMCs obtained from 4 patients at 4 time points during one event cycle and from 8 healthy children. At the febrile phase, patients exhibited hyper-inflammatory signatures in 3 monocyte clusters, including S100A12-low classical monocyte, non-classical monocyte, and S100A12-high classical monocyte, among total 21 clusters compared to the non-febrile phase of patients and healthy children. These three clusters had upregulation of gene sets associated with interferon (IFN) response. By steroid treatment, IFN response pathway was downregulated in the three monocyte clusters and CD16high NK cell cluster, suggesting that steroid treatment reduces IFN responses. Just before the febrile phase, patients exhibited increased expression of gene sets associated with response to GM-CSF. Taken together, our single-cell transcriptomic study identified that monocytes play a key role in the regularly periodic manifestation of PFAPA and are targets of therapeutic agents.
Single-cell analysis using Imaging Mass Cytometry spatially resolves immune activity in neurodegenerative brain pathology
Authors: Motta Vinicius , Zabinyakov Nick , Raza Qanber , Pfister Thomas D , Howell David , Parsotam Nikesh , Lim Liang , Loh Christina
Published: November 2024

Immune response plays a significant role in the progression of neurodegenerative diseases (NDs) such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and multiple sclerosis (MS). It is paramount to quantify the immune and structural spatial microenvironment of ND brain using research approaches that resolve tissue at a single-cell level. Imaging Mass Cytometry™ (IMC™) is a spatial biology technique that provides quantitative phenotypic evaluation of 40-plus markers.

We report the application of two new IMC imaging modes with 40-marker panels for ND research. Tissue mode (TM) was applied to perform a scan of the entire tissue followed by pixel-based analysis (PBA) to uncover spatial distribution of relevant markers. Preview mode (PM) was applied to sample the tissue at low resolution, improving informed selection of areas for high-resolution cell mode (CM) imaging on the same slide.

Using TM with PBA, we quantified the spatial distribution of immune cells in tissue compartments of ND tissues. PM and CM highlighted MS lesions devoid of myelinization and a network of α-synuclein in PD tissue. Single-cell analysis identified heterogeneity of amyloid precursor protein aggregates in AD that encompassed total and phosphorylated species of tau and phosphorylated α-synuclein. Overall, whole slide imaging techniques using IMC provide quantitative immunological and structural insights into the spatial biology of ND.

For Research Use Only. Not for use in diagnostic procedures.
Metabolism of Inflammatory T helper 17 cells from Type 2 Diabetic Patients
Authors: Ujagar Naveena , Sriram Trupti , Nicholas Dequina
Published: November 2024

Around 90% of the 537 million global diabetic adults suffer from type 2 diabetes (T2D), a metabolic disorder marked by heightened blood glucose due to insulin resistance or inadequate insulin production. T2D onset involves chronic systemic inflammation, predominantly influenced by T-helper 17 (Th17) cells. In T2D patients, defective fatty acid metabolic pathways contribute to Th17-driven inflammation. While lipid metabolism's role is established in in vitro Th17 cells, in vivo differentiated Th17 cells remain underexplored due to their low abundance. We addressed this challenge with a novel Th17 cell enrichment assay, capturing CD4+ T cells and magnetically purifying them based on IL-17A/17F/21 secretion for scRNAseq. From this transcriptomic data we established that the subsets of Th17 cells found in mice - pathogenic and non-pathogenic, are also present in humans, and that in vivo differentiated Th17 cells have a unique lipid metabolic phenotype from in vitro differentiated Th17 cells. We also characterized the metabolic state of these Th17 cells by utilizing the metabolic modeling algorithm Compass. Overlaying scRNAseq data from diabetic and control patients, we identified key lipid pathways driving Th17 inflammation in T2D. This pioneering study on endogenous Th17 cells in healthy and T2D patients, coupled with functional validation, unveils potential targets for developing immunotherapy for T2D.
TL1A drives Innate lymphoid cell-mediated granulopoiesis and colitis associated cancer
Authors: Sarmento Pires Silvia , Yang Wei , Loius Cynthia , Tran Nancy , Hassan-Zahraee Mina , Ye Zhan , Hyde Craig , Hung Kenneth , Ng Charles , Grier Alexander , Paddock Lucy , Cheing Jin , Live cell bank JRI , Targan Stephan , Niec Rachel , Josefowicz Steven , Dow Lukas
Published: November 2024

Inflammatory changes play an important role in tumorigenesis, and patients with chronic inflammatory bowel disease (IBD) are at an increased risk of developing colitis-associated cancer (CAC). Variants in TNFSF15, the gene that encodes TL1A, are associated with more aggressive IBD and advanced CRC. Here, we show that TL1A signaling is required for colitis-associated tumorigenesis and sufficient to promote CRC in mouse models. TL1A signaling in group 3 innate lymphoid cells (ILC3) promotes GM-CSF dependent activation of colonic neutrophils and colitis-associated tumors in mouse models. TL1A-mediated ILC3-dependent tumor-associated neutrophil (TAN) signatures were evident in mouse models and reduced in subjects with UC treated with anti-TL1A therapy. Furthermore, TL1A promoted ILC-dependent emergency granulopoiesis, which is similarly evident in IBD subjects with TNFSF15 risk variants. These results reveal a new mechanism by which TL1A signaling in tissue resident ILCs shape TANs and emergency granulopoiesis with the potential to guide emerging anti-TL1A therapies for IBD.
The role of 12/15-Lipoxygenase in myeloid cells during allergic fungal asthma
Authors: Makullah Mgayya , Ellis Diandra , Jones MaryJane , Steele Chad
Published: November 2024

Immunopathogenic mediators derived from the metabolism of arachidonic acid by lipoxygenases are often elevated in severe asthma. Using bone marrow chimeras, we previously reported that 12/15-lipoxygenase (12/15-LOX; Alox15) activity, specifically in hematopoietic cells, negatively contributes to airway hyperreactivity (AHR) during allergic fungal asthma. This warranted investigation into the role of 12/15-LOX in specific hematopoietic cell types during fungal asthma. CD11c is widely expressed on lung dendritic cells and alveolar macrophages. Surprisingly, deletion of 12/15-LOX in these cell types via Cd11c-Cre/Alox15fl/fl mice revealed no changes in AHR. In contrast, deletion of 12/15-LOX in neutrophils via Mrp8-Cre/Alox15fl/fl mice resulted in a small but significant reduction in AHR, suggesting that 12/15-LOX expressed by neutrophils drives immunopathogenesis during allergic fungal asthma. We further show that lungs from allergic Mrp8-Cre/Alox15fl/fl mice have a reduction in the total number of neutrophils, dendritic cells and gamma delta T cells compared to Mrp8-Cre controls. Changes in cellularity did not correlate with lung cell production of type 1, type 2 or type 17 cytokines, although specific growth factors and chemokines were lower in Mrp8-Cre/Alox15fl/fl mice. These results suggest that absence of 12/15-LOX in neutrophils is sufficient for regulating the magnitude of AHR during allergic fungal asthma, which correlated with cellular changes in the lung.
Immunology education in post-pandemic times-Hangover effects!
Authors: House Monique , Graefen Bahar , Raihan MD Obayed , Qasmieh Samah , Faridi Hafeez , Fazal Nadeem
Published: November 2024

Pandemic brought a different perspective to all ways of lives of all the people, and this included “Teachers” and “Learners” in educational world including Immunology education. We were also challenged with keeping the functional operations of our student organization alive during and after pandemic. The mission of our student chapter organization is to enhance Immunology education and encourage students to make an impact and showcase cutting science in our university. Our Facebook page during the pandemic attracted enormous visits not only by our own University students but national and even international visitors. Our Immunology student organization chapter worked in collaboration with the American Society for Microbiology (ASM) chapter during fall and spring semester. We organized tutorials, workshops, and virtual laboratories for the students. This was taken as an experiment and our data suggests that student learning, motivation, interest in immunology and educational outcomes were improved. The assessment results reported by Professor of Immunology suggest that both objective, as well as subjective educational and academic performance increased by 50% as compared to the same course taught last year. According to our data we suggest as Immunology education continues to embrace online pace, it is now more important than ever to engage with students via the social media platforms and it is well-known fact that the newer generation of students consume that the most.
“Opsonins as sugar sprinkles and B cells as music DJs?”. Effective examples of analogies used as a pedagogical tool in immunology education.
Authors: Ghittoni Raffaella
Published: November 2024

Understanding complex biological processes at the molecular level is a significant challenge for the general public with potential huge societal benefit. It also represents a crucial struggle for undergraduate students approaching immunology for the first time.

Associating complex concepts with more familiar ones, known as analogy, has been shown by several studies to be a valid educational tool used to simplify and visualize complex immunological concepts. Here, I present two effective examples of analogies, used to explain the specific immunological processes of “opsonization” and “somatic hypermutation and affinity maturation”. I will show that the introduction of analogies, following the theoretical explanation of those processes, avoids misconception and contributes in boosting student engagement, thus improving understanding and promoting the storage in permanent memory of the concepts discussed in class. Finally, I will elucidate that the use of multimedia (e.g., video and audio) to present familiar similarities stimulates students’ creativity and prompts them to think about additional examples, which likely helps yield a deeper understanding and better retention of the presented process.
Conditional MHC class I deletion in Tmem119+ microglia attenuates the brain infiltrating virus antigen specific CD8 T cell response.
Authors: Seady Marina , Maynes Mark , Hassani Asma , Thelwell Javonte , Owens Carley , Hansen Michael , Jin Fang , Johnson Aaron
Published: November 2024

Microglia activation is a hallmark feature of the immune response associated with neurological disease. In neuropathological conditions, microglia become fully competent antigen presenting cells and are proposed to perpetuate T cell activation in the brain. Historically, it has been difficult to define the extent microglia enhance CD8 T cell responses in the brain through MHC class I restricted antigen presentation. Using a novel single MHC Class I Cre Lox approach developed by our laboratory in which H-2Kb can be deactivated in Tmem119+ cells, we were able to determine the role of microglial MHC class I following intracranial (i.c.) administration of Theiler’s murine encephalomyelitis virus (TMEV) encoding the model antigen OVA. After confirming the successful deletion of H-2Kb in Tmem119+ cells using spectral flow cytometry, we investigated antigen-specific CD8 T cell expansion and the response of other immune cells in the brain and blood at 7 days post i.c. TMEV-OVA infection. We observed a marked reduction in Kb:OVA epitope specific CD8 T cells in the brain in Tmem119-cre Kb cKO mice compared to Cre- controls. This reduction in in Kb:OVA epitope specific CD8 T cells was not observed outside the CNS. Our results support a role for microglia MHC class I restricted antigen presentation for complete T cell expansion in the brain, implicating this process in protective and pathogenic neurologic conditions.
Isotype-Specific Autoantibody Signatures in Cancer and Sepsis Samples through Multiplex Profiling
Authors: Gilliam Brooke , Pfeifer Justin , McDonald John , Victorino Francisco , Qiang Xiao , Hoberg James , Lie Wen-Rong
Published: November 2024

Many diseases can be characterized by specific autoantibodies. The Luminex™ multi-analyte profiling platform enables simultaneous detection a panel of autoantibodies in a single assay with minimal sample volume. MILLIPLEX™ multiplex assays include the 15-plex Human Cancer Autoantibody Panel, 23-plex Human Cytokine Autoantibody Panel, and 20-plex Human Autoimmune Autoantibody Panel, allowing qualitative profiling of autoantibodies in serum and plasma samples. In this study, we focused on detecting cancer-associated autoantibodies and anti-cytokine autoantibodies in breast cancer, colorectal cancer, sepsis, and healthy control samples (n ≥ 20 in each group). We used the Luminex™ 200™ system and the xMAP™ INTELLIFLEX DRSE instrument for analysis. Preliminary data revealed anti-cytokine IgG autoantibodies against multiple cytokines (e.g., IL-2, IL-4, IFNγ) in cancer, sepsis, and healthy individuals. Additionally, certain cytokine autoantibodies (e.g., IL-1α, IL-12p40, IL-6) were detected in the tested disease samples but not in healthy controls. Isotype profiling demonstrated increased detection of cancer-associated autoantibodies by combining IgG and IgM signatures in cancer samples (e.g., p53, CCNB1/Cyclin B1, SOX2, CTAG1B/NY-ESO-1, Her2/ErbB2). We believe this robust MILLIPLEX™ autoantibody detection and comprehensive isotype profiling can guide further research on autoantibodies as translational biomarkers in a clinical setting.
Transient Therapeutic Inhibition of Multiple Costimulatory Molecules Modulates Persistence of Lung Tissue-resident Memory T cells
Authors: Sethi Gurupreet , Logandha Ramamoorthy Premlal Ashmitaa , Croft Michael
Published: November 2024

Costimulatory molecules, crucial for priming of T cells, development of T cell memory, and establishment of inflammatory diseases, have an unknown role in prolonged survival of Tissue-resident memory T (Trm) cells and the maintenance of relapsing inflammation. Single-cell RNA-seq data from asthmatic lungs revealed high expression of OX40, CD30L, and ICOS on CD4 T cells, aligning with transcriptomic analysis of allergen-reactive mouse CD4 Trm cells in a relapsing asthma model. Accordingly, the transient blockade of ICOSL alone or CD30L and OX40L together during tertiary allergen inhalation effectively reduced the expansion of CD44hiCD62LloCD4+ lung Trm by 40-50%, further reducing to 80-90% when concurrently targeting all three molecules and corresponding to dramatically decreased lung inflammation. Administering FTY720 with the blocking antibodies highlighted costimulatory molecules govern reactivation of both lung Trm and circulatory memory T cells that contribute to the maintenance of Trm pool. Costimulation blockade limited Trm proliferation and their continued responsiveness upon future allergen insults, explaining improved lung function. Dual blockade of ICOSL with OX40L, or ICOSL with CD30L, reproduced the effects of blocking all molecules together in limiting persistence of high numbers of functional Trm, suggesting that targeting ICOS signaling along with CD30 or OX40 signaling holds promise as a clinical treatment for various immune-mediated inflammatory diseases
Neurogenic vasoactive intestinal peptide alters tcr proximal signaling in naive t cells
Authors: Adebamowo Adedola , Rosenberg Kenneth , Singh Nevil
Published: November 2024

Communication between the nervous and immune systems is critical for regulating the immune response. Neuropeptides have been observed to exert modulatory effects on many different lymphocyte subsets. In mouse models, we found peripheral T cells expressed a set of 26 neurotransmitter receptors and exhibited distinct patterns across T cell subsets. The Vasoactive Intestinal Peptide receptor (VIPR1) had an intriguing pattern with high expression in naïve peripheral CD4 T cells and none in Tregs. Upon T cell receptor (TCR) stimulation, VIPR1 expression rapidly declines, suggesting endogenous VIP primarily affects resting naïve T cells. Functionally, treatment with VIP dampened the early activation of naïve T cells, as measured by reductions in proliferative responses and IL-2 secretion in response to antigenic stimulation. Mechanistically, VIP-exposure directly affected signaling via the TCR. Although the early events in the signaling cascade, such as the phosphorylation of ZAP70 was intact, downstream activation of ERK was reduced. Endogenous VIP has a very short lifespan and therefore localized production is likely to impact naïve T cell activation. To map the cellular sources of VIP we generated strains of VIP-lineage-tracing mice. Preliminary analysis shows strong expression in multiple neurons. These data suggest that VIP secreted by neurons has the potential to influence T cell response in a fairly broad manner at the early stage of an immune response.
Formyl Peptide Receptor 1 regulates early control of Mycobacterium tuberculosis infection.
Authors: Nafiz Tanvir Noor , Saqib Mohd , Sankar Poornima , Mishra Bibhuti
Published: November 2024

Formyl peptide receptors (FPR) are playing a pivotal role in phagocyte migration in response to bacterial and host-derived chemoattractants. Despite their established roles, their involvement in immunity to tuberculosis remains unexplored.

In this study, we investigated the contributions of Fpr1 and Fpr2 to host defense against Mycobacterium tuberculosis (Mtb) infection. Fpr1 and Fpr2 expression increased in the lungs of rabbits as early as 3 hours and remained elevated during the chronic phase of infection. Additionally, these receptors were increasingly expressed in neutrophils of mice highly susceptible to infection, implying a potential role in the host response to Mtb.

We assessed bacterial burden, lung damage, and cellular inflammation following infection of Fpr1-/- and Fpr2-/- mice with a hypervirulent W-Beijing lineage strain of Mtb. While Fpr2 deletion exhibited no discernible impact on host resistance, Fpr1 deficiency compromised bacterial control in the lungs. This effect was particularly pronounced in genetically susceptible mice treated with Fpr1-specific inhibitors. In vitro experiments demonstrated that bone marrow–derived neutrophils and macrophages lacking Fpr1 were incapable of controlling intracellular Mtb growth.

Collectively, our findings underscore the differential roles of Fpr1 and Fpr2 in the host defense against Mtb infection, highlighting Fpr1 as a potential therapeutic target for enhancing immune responses against tuberculosis.
FIP200 promotes Mycobacterium tuberculosis pathogenesis independent of its role in autophagy
Authors: Dubey Neha , Woodson Reilly , Kinsella Rachel , McNehlan Michael , Naik Sumanta , Kreamalmeyer Darren , Stallings Christina
Published: November 2024

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), remains a critical health issue. Xenophagy, the targeting of Mtb to autophagosomes within infected cells is ineffective at controlling Mtb replication in vivo. We discovered that autophagy-associated proteins function outside of xenophagy to impact Mtb pathogenesis. We reveal a distinct autophagy-independent function of FIP200 in CD11c+ alveolar macrophages (AMs) to control Mtb burden in mice lungs at 7 days post-infection. Intra-tracheal airway labelling shows that despite the abundance of inflammatory cells, including AMs, in airway and interstitium, airway resident AMs precisely exhibit better infection control in Fip200fl/fl-LysM-Cre mice lungs.FIP200, beyond its role in autophagy, is shown to regulate signaling pathways to suppress TNF-induced apoptosis and promote cell survival. Compared to flox control, loss of Fip200 resulted in significantly higher apoptosis in AMs, in the lungs of naïve mice, which is sustained during infection. We also observed higher reactive oxygen species production by the AMs in lungs. This observation extends to Streptococcus pneumoniae infection, where loss of FIP200 in CD11c+ AMs caused similar increase in apoptosis and ROS,with reduced bacterial burden in mouse lungs. Thus, we propose a model where the FIP200 inhibits the apoptosis and ROS production in AMs, which impairs the antimicrobial defense in these cells, thus promoting Mtb infection in the airways following infection.
Modification of Staphylococcal aureus quorum sensing and virulence factor expression by cold plasma treatment
Authors: Melvage Autumn , Smith Carly , Connelly Amanda , Baldomero Genevieve , Lucas Abby , Watkins Amanda , Hickok Noreen , Freeman Theresa
Published: November 2024

Staphylococcal aureus (S. aureus) resides in biofilms and produces virulence factors. Cold atmospheric plasma treatment can be used to kill and reduce bacteria virulence expression. This study will observe the effects of cold plasma treatment on S. aureus killing, biofilm disruption, virulence factor, and quorum sensing gene expression in vitro and in vivo.

S. aureus biofilms were grown in vitro on discs and then treated with plasma (J-Plasma; Apyx Medical Inc.) CFU counts, Crystal Violet Staining, and SEM were conducted to determine biofilm disruption and killing. Gene expression was determined by qPCR for Superoxide dismutase (SodA), Panton-Valentine Leukocidin (luk-PV), alpha-hemolysin (Hla), and AgrB and SarA. A similar study was performed on muscles infected in a rat model, treated and untreated, at Days 0, 4 and 14.

Cold plasma-treated biofilms on discs had significantly less bacterial CFU counts, lower crystal violet absorbance, and visible disturbances of biofilm. qPCR showed a significant reduction of virulence factor and quorum signaling genes. In vivo, all day 4 and 14 muscle samples had significantly higher CFU counts than 6-hour samples. Results showed a fold change reduction after plasma treatment in virulence and quorum sensing gene expression.

Cold plasma treatment disrupts S. aureus biofilm formation and gene expression, both in vivo and in vitro, highlighting the potential of cold plasma treatment to aid in the clearance of S. aureus infections.
Generation of iPSC derived CD19 CAR iNK cells for treatment of autoimmune disorders
Authors: Walsh Ariane , Grandolfo Davide , Pichery Mélanie , Turfkruyer-Housson Mathilde , Richter Mandy , Hublitz Philip , Esquerré Michael , Bombarde Oriane , Holtzinger Audrey , Scheel Andreas , Wagner Nadja , Cernecka Hana
Published: November 2024

Treatment of autoimmune diseases (ADs) relies on broad immunosuppressive agents that are often not curative. Autologous CAR T cell approaches demonstrated disease remission in patients with Systemic lupus erythematosus (SLE). Among cellular abnormalities typical for ADs, NK cells dysfunction was reported. We believe that using iPSC derived CD19 CAR NK cells (iNK cells) to eliminate disease-causing circulating plasma cells could be a novel therapeutic approach for ADs mediated by B cells. Evotec’s validated GMP iPSC line was genetically modified by a knock-in of a CD19-CAR and differentiated into iNK cells using a feeder-free 3D differentiation process. Successful knock-in was confirmed by flow cytometry. Expression of the CAR did not negatively impact the differentiation of the iNK cells. The CD19 CAR iNK cell phenotype and scRNA sequencing profiles are comparable to blood-derived NK cells. To evaluate the ability of CD19 CAR iNK cells to selectively eliminate primary B cells, patient-derived B cell chronic lymphatic leukemia (CLL) cells and primary B cells from healthy donors were co-cultured with CAR iNK cells. Compared to wildtype (WT) iNK cells, CD19 CAR iNK showed specific killing of CD19-positive CLL cells and primary B cells. iPSC-derived CD19-CAR NK cells show increased cytotoxicity and specific targeting of CD19 positive primary patient and healthy subject derived B cells, demonstrating their potential suitability for the treatment of ADs such as SLE.
C57BL/6J-Trex1em3Lutzy/J KO mouse as a type 1 IFN dependent model for SLE preclinical drug discovery
Authors: Case Laure , Presa Max , Tata Saurabh , Martin Paige , Tracey Katelyn , Lutz Cat
Published: November 2024

Systemic Lupus Erythematosus is a chronic autoimmune disease characterized by a broad spectrum of clinical manifestations. An important role of the type I interferon (IFN) pathway has been identified in SLE patients. No one mouse model completely recapitulates the complexity of the human disease. Nonetheless, mouse models of SLE have been instrumental in our understanding of the genetic and cellular requirements for SLE induction and represent a valuable resource for testing SLE therapies and drug candidates in vivo. Here we present preclinical reference data in a Trex1KO mouse model generated at the Jackson Laboratory using CRISPR technology. Trex1KO mice have a median survival of approximately 165 – 185 days. Body weight is significantly reduced in both male and female KO versus WT. Unlike NZBWF1/J and MRL/MpJ mouse models that develop lupus nephritis, the Trex1 KO develops dilated cardiomyopathy due to leukocyte infiltration and cardiomyocyte degeneration. Here we treated KO mice with anti-IFNα antibodies through 32 weeks of age. We observed a significant treatment effect on survival, reduction in leukocyte infiltration and degeneration of the heart, and percentage of T peripheral helper T cells in spleen. A mouse model recapitulating a role for type I IFN in disease pathogenesis has been lacking in the common models of SLE, and these data indicate that the Trex1KO model is relevant for the screening of targeted therapies of type I IFN and related pathways.
Vasoactive Intestinal Polypeptide in the Enteric Neural Plexus Promotes Microbiome Diversity and Resistance to GvHD
Authors: Li Yiwen , Wang Shuhua , Zhang Hanwen , Liu Yutong , Passang Tensin , Giver Cynthia , Waller Edmund
Published: November 2024

Graft-versus-host disease (GvHD) in allo-transplant recipients is characterized by donor T cell- mediated damage to recipient organs and changes in the gut microbiome. Vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, directs T cell responses towards a Th2 phenotype while inhibiting proinflammatory cytokine production. VIP-producing enteric neurons contribute to the mucosal barrier and maintain gut microbiome homeostasis. The impact of VIP production by host cells on GvHD remains unclear. We examined the effect of endogenous VIP synthesis in modulating GvHD in multiple murine allo-BMT models. VIP knock-out mice were exquisitely sensitive to GvHD, experiencing activation and expansion of Th1- and Th17-polarized T cells and early death. Transplanting radiation chimeric mice in which the VIP gene was knocked out in either hematopoietic or non-hematopoietic cells demonstrated that VIP production by non-hematopoietic tissues limited the GvHD activity of donor T cells. Immunofluorescence staining demonstrated VIP expression in enteric neurons in allo-BMT recipients, consistent with local effects of VIP on T cells and the gut microbiome. VIP- KO mice had lower gut pH, higher frequencies of Bacillota species, and less microbiome diversity post-transplantation. These data support the crucial role of VIP in maintaining the diversity of the gut microbiome with beneficial commensal species and in modulating the alloreactivity of donor T cells after allo-BMT.
Entolimod, a clinical stage flagellin derivative, improves clinically relevant chemo-immunotherapy responses in pre-clinical triple negative breast cancer
Authors: Brackett Craig , Aldrich Alyssa , Punch Patrick , Greene Kellee , Molodtsov Ivan , Gudkov Andrei , Abrams Scott
Published: November 2024

Chemotherapy+anti-PD-1 is FDA approved and has improved outcomes for locally advanced and metastatic PD-L1+ triple negative breast cancer (TNBC). Unfortunately, a need still exists to improve this chemo-immunotherapy (CTx-I) platform especially in advanced disease. Entolimod, an agonist of the TLR5 and NAIP5 receptors, is our flagellin derivative which has successfully completed Phase I safety trials. We previously showed that systemically administered entolimod enhances durable antitumor immunity against metastasis of 4T1, a preclinical PD-L1+ TNBC model. However, the antimetastatic activity of single-agent entolimod was not completely effective, highlighting the need to combine entolimod with additional immune-based modalities such as CTx-I to further improve tumor control. Here, we used Paclitaxel+anti-PD-1 as a clinically oriented CTx-I platform in pre-clinical metastatic TNBC models. CTx-I+entolimod effectively controlled primary tumors and reduced lung metastatic burden more so than the single or double agent treatments. Consistent with our prior work that neutrophils express functional TLR5, depletion abrogated CTx-I+entolimod efficacy. Collectively, the immune enhancing effects of entolimod can be leveraged to boost urgently needed CTx-I outcomes in TNBC. Because entolimod has successfully completed Phase I clinical trials, this preclinical work supports the rationale design of a clinical trial that combines CTx-I with entolimod as an effective immunotherapy platform.
Adeno-Associated Virus-Mediated IL-2 Delivery in Liver Cancer: Immune Activation and Therapeutic Challenges
Authors: Chuang Ya-Hui
Published: November 2024

Hepatocellular carcinoma (HCC), a prevalent primary liver cancer, poses a significant global health challenge. The liver's immune population is selectively enriched in natural killer (NK) cells, natural killer T (NKT) cells, and CD8+ T cells, which play critical roles in anti-tumor responses. Interleukin-2 (IL-2) holds promise in activating these immune cells for cancer therapy, but its short half-life and systemic toxicity limit its application. This study aimed to enhance hepatic NK, NKT, and T cells using a liver-preferred adeno-associated virus (AAV) to deliver IL-2 (AAV-IL-2) sustainably for HCC therapy in a syngeneic orthotopic mouse model. At first, we administered high, medium, and low doses of AAV-IL-2 to naive mice, revealing that only the medium dose expanded hepatic NK, NKT, and T cells without inducing toxicity. In HCC-bearing mice, low-dose AAV-IL-2 treatment had minimal effects. Medium-dose AAV-IL-2 treatment expanded and activated hepatic NK, NKT, and T cells but also induced intratumoral CD8+ T cell exhaustion. Notably, medium-dose AAV-IL-2 had no suppression of tumor progression but higher mortality. These results underscore the challenging balance required for effective HCC treatment with IL-2, as a medium dose successfully enhances hepatic immune cells but simultaneously induces CD8+ T cell exhaustion and higher mortality.