bluuebunny/crossref_metadata_2025 huggingface dataset:
NOX2-deficient neutrophils facilitate joint inflammation in serum-induced arthritis
Authors: Liao Yi-Chu , Shieh Chi-Chang K
Published: January 2023

Immune-mediated inflammatory arthritis, including rheumatoid arthritis (RA), is a family of chronic inflammatory diseases of joints. NADPH oxidases 2 (NOX2) complex in phagocytes generates high concentrations of ROS to kill the engulfed microbes. Inherited mutations in the subunits of NOX2 result in chronic granulomatous disease (CGD). Subjects with defective NOX2 are susceptible to recurrent infections and a variety of autoimmune conditions. According to our previous studies, NOX2-deficient mice developed more severe immune-mediated arthritis, but depletion of neutrophils suppressed the development of serum-induced arthritis in these mice. Based on the hypothesis that NOX2-deficient neutrophils play a pro-inflammatory role in immune-mediated arthritis, we investigated the activity of neutrophil from joints to facilitate inflammation. We found that there are more neutrophils accumulations in NOX2-deficient joints, compared to WT joints. Both NOX2-deficient /WT neutrophils affected the anti-CD3 and anti-CD28 induced T lymphocyte proliferation, however, NOX2-deficient neutrophils were less suppressive. Moreover, NETs formation and high level of citrullinated histone H3 were detected in joints of NOX2-deficient mice at day 3 of arthritis induction. Furthermore, neutrophils from RA joints were found to suppress the T cell proliferation. Finally, carbonylated protein and total GSH level were lower in synovial fluid of RA patients when compared with those in osteoarthritis joints. Our results implicate that neutrophils may be an important cellular factor that lead to more severe arthritis in the NOX2-deficient subjects.
Kindlin-3 organizes a ring of clustered high affinity β2 integrins during human neutrophil spreading under flow
Authors: Fan Zhichao , Wen Lai , Yeh Yi-Ting , Kiosses William B , Gutierrez Edgar , Groisman Alex , Francois Joshua , Lasheras Juan C , del Alamo Juan C , Chien Shu , Ginsberg Mark H , Petrich Brian G , Ley Klaus
Published: January 2023

Neutrophils are vital for inflammation and immune defense. Dependent on β2 integrins, spherical neutrophils spread on vascular endothelium after arrest, which is critical for their recruitment from circulation to resist high shear flow and to initiate intravascular crawling. Here, we use high-resolution quantitative dynamic footprinting microscopy to monitor neutrophil spreading on a substrate of recombinant ICAM-1 and P-selectin under flow. A homogenous binding assay using the conformation-reporter antibodies mAb24 (reporting high-affinity β2, H+) and KIM127 (reporting extended β2, E+) showed three conformations of activated β2 integrins. E−H+ β2 integrins increased before E+H− and E+H+ conformations at the beginning of neutrophil spreading. Integrin extension depended on Syk-mediated integrin outside-in signaling. The ring of E−H+ and E+H+, but not E+H− β2 integrins was fully formed during late neutrophil spreading just before migration. Using kindlin-3-GFP fusion proteins, a ring of kindlin-3 was observed before the ring of H+ integrins appeared. These findings show spatially coordinated integrin activation during spreading. The previously unrecognized E−H+ conformation is the pioneer integrin for neutrophil spreading and appears to be organized by kindlin-3.
The pathogenic role of choroid plexus infiltrating T cells in Neuropsychiatric Lupus
Authors: Moore Erica , Huang Michelle , Reynolds Cara , Putterman Chaim
Published: January 2023

Neuropsychiatric systemic lupus erythematosus (NPSLE) manifests as a range of symptoms, including cognitive abnormalities, mood disorders, and psychosis. The pathogenesis is complex and not yet completely understood. We recently proposed that the choroid plexus (CP) could be an alternative entry site to the blood brain barrier for immune mediators to enter the central nervous system. In the MRL/lpr lupus mouse model, the CP is heavily infiltrated with T and B cells, which is reminiscent of human NPSLE patients whose autopsies revealed infiltrates in the CP. Nevertheless, the role of CP infiltrating T cells in the pathogenesis is not known and is the objective for this study. In two cohorts, we evaluated whether intracerebroventricularly (ICV) delivered MRL/lpr CP T cells accelerate neuropsychiatric disease in young female MRL/lpr mice. Six week old female MRL/lpr mice were injected with MRL/lpr CP CD3+ T cells, MRL/lpr splenic CD3+ T cells, or PBS. The T cells were pooled from the CP and spleen of 16 week old MRL/lpr mice and expanded with anti-CD3/CD28 antibodies (1 ug/mL) and mIL-2 (10 U/mL) for 4 days. About ~200,000 cells were injected into the lateral ventricle from the bregma at AP −0.34 mm, ML −1.0 mm, and depth −2.0 mm. After 4 weeks post-injection, we found that CP T cell recipient mice had increased cognitive deficits compared to PBS controls. Histological assessment revealed an increase in lymphocytic infiltration, the number of proliferating cells, and IgG and complement deposition, in the choroid plexus of CP T recipient mice. Our result indicate that T cells can migrate to and proliferate in the CP following ICV injection, and that brain infiltrating MRL/lpr T cells may be a key contributor to the pathogenesis of NPSLE.
Oxysterols induce proinflammatory response via Focal Adhesion Kinase (FAK) signaling
Authors: Casimiro Kim Roxana Chiok , Pokharel Swechha Mainali , Bose Santanu
Published: January 2023

We recently reported that the oxysterol 25-hydroxycholesterol (25HC), which is an oxygenated form of cholesterol, promotes proinflammatory response via Integrin-Focal Adhesion Kinase (FAK) signaling. However, it is unknown whether other non-25HC oxysterols possess similar proinflammatory activity induced by FAK signaling pathway. Thus, we tested two non-25HC oxysterols, 22-hydroxycholesterol (22HC) and 7-alphahydroxycholesterol (7-alpha-HC), for their ability to induce proinflammatory response in macrophages. Both of these oxysterols induced a proinflammatory response since we observed production of the proinflammatory cytokine tumor necrosis factor alpha (TNF) from macrophages treated with 22HC and 7-alpha-HC. Furthermore, this response was mediated via FAK signaling as TNF production from 22HC treated macrophages was abrogated in the presence of a FAK-inhibitor. Taken together, this study suggests that non-25HC oxysterols can utilize a similar signaling pathway as 25HC to induce proinflammatory response. Studies are currently in progress to dissect the molecular mechanisms involved in triggering proinflammatory response by 22HC and 7-alpha-HC.
Follicular dendritic cell migration is facilitated by the expression of matrix metalloproteinase 3 through TNFα-ERK1/2-AP-1 signaling
Authors: Pak Hyo-Kyung , Kim Yong-Woo , Lee A-Neum , Roh Jin , Chung Yoo-Sam , Park Chan-Sik
Published: January 2023

Folliculardendritic cells (FDCs) are the crucial stromal components in the germinal center and have the pivotal roles in high-affinity antibody production. FDCs are developed and maintained in the germinal centers by lymphotoxin α1/β2 and tumor necrosis factor-α (TNFα). However, many of the features of FDCs are still not identified, particularly its migration. Here, we demonstrate for the first time that the migration of human FDCs is enhanced by TNFα-mediated metalloproteinase 3 (MMP3) expression. MMP3 was highly expressed in the normal human germinal centers. MMP3 expression was markedly increased in human primary FDCs by TNFα. TNFα induced the ERK1/2 phosphorylation and the promoter activity of the AP-1 binding sites in the Mmp3 promoter region. TNFα treatment increased FDC migration and the knockdown of MMP3 significantly reduced the TNFα-induced migration of FDCs. Overall, we identified a novel control mechanism for the expression of MMP3 in FDCs to modulate the migration, which may play an important role in lymphoid follicle formation. Since lymphoid follicles are observed in the lesions of autoimmune diseases and lymphomas, controlling the MMP3/TNFα-mediated migration of FDCs can provide novel insight into therapeutic modalities for aberrant germinal center-associated disorders.
CD103 promotes the proinflammatory response of gastric resident CD4+ T cell in Helicobacter pylori- positive gastritis
Authors: Wu Yongjian , Ming Siqi , Liang Siping , Wu Minhao , Chen Peiyu , Geng Lanlan , Gong Sitang
Published: January 2023

As an integrin found on T cell residence, CD103 is associated with the CD8+ T cell response in allogeneic transplant rejection, autoimmune diseases and cancer, but its role in infection and inflammation remains unclear. In this study, biopsy specimens of the gastric mucosa were obtained from patients with H. pylori-positive gastritis. We found that CD103 was highly expressed in CD4+T cells of the gastric mucosa from patients with H. pylori-positive gastritis. Mucosal resident CD103+CD4+T cells exhibited an increase in the CD45RO+CCR7− effector memory phenotype and high expression of the chemokines CXCR3 and CCR9 compared with those in CD103−CD4+ T cells. An In vitro coculture study demonstrated that H. pylori-specific antigen CagA/ VacA-primed dendritic cells (DCs) induced proliferation as well as IFN-γ, TNF and IL-17 production by CD103+CD4+ T cells from patients with H. pylori-positive gastritis. Furthermore, blocking CD103 with a neutralizing antibody reduced proliferation and IFN-g, TNF and IL-17 production by CD103+CD4+ T cells cocultured with DCs. Moreover, immunoprecipitation revealed that CD103 interacted with TCR α/β and CD3ζ, and activating CD103 enhanced the phosphorylation of ZAP70 induced by the TCR signal. Finally, increased T-bet and Blimp1 levels were also observed in CD103+CD4+ T cells, while activating CD103 increased T-bet and Blimp1 expression in CD4+ T cells. Our results explored the function of CD103 in gastric T cells from patients with H. pylori-positive gastritis, which may provide a therapeutic target for the treatment of gastritis
Mechanism of Microvascular Endothelial Cells Protected by Pulsatillae Decoction Activated Neutrophils to Kill Bacteria
Authors: He shangwen , Liu Xiaoye , Li Qiuyue , Zhang Hui , Hou Silu , Xu Yuanyuan , Dong Hong
Published: January 2023

Neutrophils play critical a role in bacterial infection and tumor immunity. The migration of neutrophils across microvascular endothelial cells has been a focus of increased interest within the past decade. A traditional Chinese herbal formula, Pulsatillae Decoction (PD), has the effects of improving the permeability of MVECs, inhibiting apoptosis and enhancing cell activity. But there are very few studies linking the three, and the effect of PD on the bactericidal function of PMNs transendothelial migration is unclear. To investigate the protective effect of PD on the damage of Rat microvascular endothelial cells (RIMVECs) induced by LPS and it’s mechanism of activating neutrophils (PMNs) to kill bacteria by protecting RIMVECs, we established a model of LPS-induced RIMVECs injury and to evaluate the protection of PD against LPS-induced RIMVECs injury. We observed that LPS-injured RIMVECs inhibited the extracellular and intracellular bactericidal activity of transendothelial neutrophils, PD-protected RIMVECs promoted the extracellular and intracellular killing activity of transendothelial neutrophils and significantly increased NE and CG concentration in transendothelial neutrophils. In addition, SWATH/DIA proteome results showed that 11 consensus proteins were down-regulated in the model group and up-regulated in the treatment group, 23 consensus proteins were up-regulated in the model group and down-regulated in the treatment group. PD was likely to protect RIMVECs and activate the sterilization of PMNs by regulating these differential proteins.
CXCL10+ perivascular clusters nucleate Th1 cell tissue entry and activation in the inflamed skin
Authors: Prizant Hen , Patil Nilesh , Negatu Seble , Livingston Alexandra , Leddon Scott , Luster Andrew D , Fowell Deborah J
Published: January 2023

Efficient recruitment and correct positioning of T cells within infected peripheral tissues are crucial for pathogen clearance. Using a dual-chemokine reporter (Groom et Al. Immunity, 2012) mouse and intra-vital imaging, we identified a preferred tissue entry and accumulation site for effector Th1 cells, defined by clusters of CXCL9 and CXCL10 expressing hematopoietic cells, enriched with MHC-class-II+ cells, in restricted perivascular spaces within the inflamed dermis. Unbiased computational analysis of motility dynamics of antigen-specific Th1 cells within these clusters, revealed cell confinement characteristics. CXCR3 on Th1 cells and presence of cognate antigen were critical for persistence within clusters and T cell activation. Newly recruited Th1 cells strongly amplified CXCL9 and CXCL10 expression, mainly within skin monocyte-derived dendritic cells, in an antigen- and interferon-gamma-dependent manner, resulting in augmented chemokine clusters. Our data suggest a CXCR3-mediated mechanism of intrinsic amplification loop for optimal Th1 cell recruitment, positioning and activation in restricted antigen presentation sites within the inflamed skin.
Chemical modification strategies for photo-induced control of immune cell signaling
Authors: Perdue Lacey A , Do Priscilla , Chyong Andrew , Costanza Emma , Kim Hye Ryong , David Camille , Kellner Anna , Salaita Khalid , Lesinski Gregory , Porter Christopher C. , Dreaden Erik C.
Published: January 2023

Cytokine therapies show great potential to induce cancer immune elimination and have demonstrated therapeutic benefit in a subset of patients. However, controlling the pleotropic effects of these immune signal effectors is a challenge. Here, we present an approach to enable optical control of cytokine activity by caging the activity of these proteins via labeling with wavelength-specific, photo-labile polymers (i.e. photo-caging). We create a system to measure light-induced polymer cleavage (uncaging) through fluorescence dequenching and establish: high wavelength-selectivity, protein activation through tissue mimics, and micrometer-scale uncaging resolution (via photopatterning). We focus on cytokines IL-2, IL-12 and IL-15 and demonstrate ability to reversibly modulate cytokine size and activity. We show photo-caging increases cytokine size by up to 20 fold, resulting in increased serum half-life. Additionally, cytokine activity (via T-cell proliferation) is blunted by 1,000 fold and near fully recovered through light-induced uncaging. Affinity measurements demonstrate photo-caging disrupts the bias of IL-2 towards IL-2Rα expressed on immunosuppressive T-regulatory cells. Further, photo-caging negates the ability of IL-2 to increase antigen specific (OT-1) cell activation, but is restored upon uncaging. In summary, we present the synthesis and therapeutic potential of these optically activated cytokines. In future studies we aim to use cytokine photo-caging as a means to provide new insights into the mechanics of cancer immune elimination and achieve tissue exclusive activation by exploiting spatially and temporally constraining cytokine activation in chip-based organoids and in vivo cancer models.
Inflammation impairs IL-2R signaling in regulatory T cells
Authors: Hsiung Sunnie , Malek Thomas R
Published: January 2023

Low-dose IL-2 is a promising therapy to boost Tregs in autoimmune diseases. Inflammation accompanying autoimmunity destabilizes Tregs to diminish their function. Here we explored how the extent of acute inflammation induced by LPS and poly(I:C) and of chronic inflammation induced by immunization with MOG and pertussis toxin (MOG/PT) affects IL-2R signaling in Tregs. IL-2-dependent pSTAT5 was significantly, but transiently, reduced in Tregs ex vivo from C57BL/6 mice exposed to LPS and MOG/PT, but not poly(I:C). LPS and PT, but not poly(I:C), induced inflammation through TLR4. IL-2-dependent pSTAT5 was also decreased in WT mice treated with only PT or in TLR4 Treg-conditional knockout mice treated with LPS. Thus, impaired IL-2R signaling is likely dependent on monocyte-derived inflammatory mediators through TLR4 signaling. LPS also decreased Treg proliferation as measured by Ki67 and increased CD69 expression. In contrast, these changes were not seen after treatment with poly(I:C) and MOG/PT, consistent with inflammation affecting Tregs independent of lower IL-2R signaling. When Tregs from LPS-treated mice were stimulated with IL-2 in vitro, pSTAT5 was induced, indicating that Tregs are not intrinsically unable to respond to IL-2. RNAseq of Tregs from LPS-treated mice revealed substantial changes in their transcriptome, suggesting a potential complex mechanism that impairs IL-2R signaling and other activities in Tregs in vivo. Our data support a model where inflammation lowers IL-2R signaling in Tregs by decreased IL-2 in the Treg microenvironment and causes other induced changes in Tregs to lower their function. Thus, a beneficial feature of low-dose IL-2 therapy may be to counteract negative effects of inflammation on Tregs.
IL-9 Represses Intestinal Granzyme A Positive CD4 T Cells in Mice with Allergic Airway Inflammation
Authors: Hayes Tristan A , Ulrich Benjamin J , Fu Yongyao , Pajulas Abigail , Kaplan Mark H
Published: January 2023

Mediated by immune responses in the airways, symptoms of allergic airway diseases include shortness of breath, difficulty breathing, and wheezing. Responses in distant tissues, such as increased lymphocyte proliferation and cytokine secretion, are also exhibited. How airway sensitization mediates these distant effects is unclear. We recently described the development and function of granzyme A-expressing T cells that control intestinal inflammation. Using models of chronic and memory allergic airway inflammation, we now show that a granzyme A expressing CD4 T cell population in the intestine increases during these diseases. This response is amplified in mice that lack IL-9 signaling through a deficiency in IL-9 receptor levels (IL9RKO) or receive an IL-9 antibody blockade. In a chronic intranasal allergen challenge model, we saw elevated levels of granzyme A expressing CD4 T cells in IL9RKO mice when compared to wild-type controls. In a memory internasal allergen model using wild-type mice (chronic allergen exposure, followed by an allergen-free rest period, and an allergen re-exposure), granzyme A expressing cells in the intestine were increased upon IL-9 antibody blockade. Blocking IL-9 or administering IL-9 to naïve mice or cell cultures that generate granzyme A expressing T cells did not affect cell prevalence. Therefore, concurrent allergic airway inflammation seems to be required for the proliferation of these granzyme A expressing T cells. Together, these studies suggest that allergic airway inflammation increases granzyme A expressing CD4 T cells in the intestine and that IL-9 attenuates this effect. Moreover, these results show that allergic airway inflammation impacts immunity at a distant mucosal site.
Autocrine and paracrine IL-2 signals collaborate to regulate distinct phases of CD8 T cell memory
Authors: Kalia Vandana , Yuzefpolskiy Yevgeniy , Vegaraju Adithya , Smith Kendall A , Sarkar Surojit
Published: January 2023

Differential IL-2 signaling is implicated in memory CD8 T cell fates. Autocrine IL-2 production is a hallmark of polyfunctional, long-lived lymphoid memory cells. Whether the IL-2 signals perceived by CD8 T cells come from autocrine or paracrine sources, the timing of these signals, and their differential impact on the qualitative outcomes of CD8 T cell responses are not clearly understood. We used two distinct models of germline and conditional autocrine IL-2 ablation in post-thymic CD8 T cells to specifically dissect the requirement and timing of autocrine IL-2. Our studies show that autocrine IL-2 is uniquely required during primary CTL responses to program the key memory property of robust recall responses. Ablation of autocrine IL-2 in fully differentiated wild type memory cells immediately prior to rechallenge did not impact their recall expansion potential. Furthermore, we found that paracrine IL-2 could effectively supplant autocrine IL-2 for primary expansion, effector differentiation and memory maintenance, but not recall expansion. Likewise, our data highlight the redundancy of autocrine and paracrine IL-2 in optimally driving equivalent metabolic programs associated with effector and memory states. While CD4 licensing of DCs is needed for optimal programming of autocrine IL-2 in CD8 T cells, we found that the requirement for CD4 help is less critical for autocrine IL-2 production in the context of inflammatory infections. These findings have implications in adoptive T cell immunotherapies where expansion of engineered cells is largely driven by paracrine IL-2, which can potentially impact their long-term sentinel function.
Interleukin 2 enhances and sustains IFNy induced STAT1 activation in subpopulations of CD4+ and CD8+ T lymphocytes in untreated relapsing remitting multiple sclerosis (RRMS) patients
Authors: Pope Brandon J , Sharma Vishal , Meador William , Reynolds Richard , Bridges S. Louis , Raman Chander
Published: January 2023

Multiple sclerosis (MS) is the most common demyelinating disorder in adults, where 85 percent of patients exhibit relapses, followed by remission (RRMS). IFNy administered to RRMS patients increases relapse rate. IFNy producing myelin specific T lymphocytes correlate with functional impairment in MS. RRMS derived neuronal lesions show increased expression of IFNy induced genes on microarray analysis, compared to controls. At time of diagnosis, we observed comparable levels of IL2 and IFNy in the plasma of RRMS patients. Since IL2 constrains differentiation of specific CD4+ effector lineages (i.e. Th17, Tfh) in the context of autoimmunity, we predicted that its expression was an effort to counteract IFNy signals. To test for this possibility, we interrogated the effect of IL2 on IFNy induced activation of STAT1 in subpopulations of CD4+ and CD8+ T cells (effector, memory, and regulatory) from newly diagnosed RRMS patients. In all CD4+ and CD8+ T cell populations from RRMS, IFNy induced activation of STAT1 that peaked at 1h followed by decay at subsequent timepoints. In a subset of RRMS patients, costimulation with IFNy and IL2 resulted in enhanced and sustained activation of STAT1 in CD4+ and CD8+ naïve and effector T cells, CD4+ memory T cells, and Treg cells. IFNy had no effect on IL2 induced STAT5 activation. From these results, we infer that in some MS patients IL2 will collaborate with IFNy to enhance effector T cell activation to promote autoimmune neuroinflammation. These results suggest that low dose IL2 therapy could exacerbate RRMS disease severity in such patients.
Active learning about the dynamic and complex nature of CD4+ T cell differentiation with computer network modeling and simulations
Authors: Helikar Tomas , Helikar Resa M , Brown Deborah M
Published: January 2023

CD4+ T cells provide cell-mediated immunity in response to various antigens. During an immune response, naïve CD4+ T cells differentiate into specialized effector T helper cells (e.g., Th-1,-2,-17, etc.), and induced regulatory (iTreg) T cells. Their proper response to pathogens is governed by complex, non-linear dynamic networks consisting of various types of cells and their associated communication mediators (cytokines). The regulation of CD4+ T cell differentiation is a major topic of many post-secondary immunology-related courses.

However, the traditional lecture and textbook-based learning format in these (and other life sciences) courses makes it challenging for students to fully conceptualize and appreciate the complex and dynamic nature of such processes. We have developed a new learning approach that enables students to build, simulate, and investigate computational models of processes embedded in biological systems. This method is facilitated through an easy-to-use software, Cell Collective (https://cellcollective.org), that makes computational modeling accessible to any student and instructor (i.e., no prior computational modeling experience is necessary). The simulation lessons have been designed to be turn-key, self-contained, and adoptable by any instructor without the need to modify their syllabus, and with a minimal learning curve. They can be used in-class, assigned as homework, as well as deployed as extensive lab investigations. Here, we will present a new modeling and simulation lesson that enables students to learn about the dynamic complexities of CD4+ T cell differentiation, such as the effects of different cytokines and their dosage on the differentiation outcome, feedback loops, etc.
Development of Curriculum Guidelines for Undergraduate Immunology Education –a Report by a Task Force on Undergraduate Immunology Curriculum Guidelines
Authors: Justement Louis B , Bruns Heather A. , Elliott Samantha , Sletten Sarah , Sparks-Thissen Rebecca , Condry Danielle , Wisenden Brian , Lal Archana , Paustian Timothy , Mixter Philip F , Taylor Rebekah , Pritchard Rachel , Vanniasinkam Thiru , Dorsam Glenn , Freier David , Pandey Sumali
Published: January 2023

Until recently, immunology education was primarily introduced at the graduate level. However, in the age of modern medicine, a fundamental understanding of the immune system is becoming increasingly critical. The immune system affects every organ system in the body, and entails many concepts that are critical for training students who wish to pursue health professions or research-intensive careers. In addition to medicine, ecologists and computer scientists have lately been attracted to the field to investigate problems related to ecoimmunology or for development of machine learning tools for disease modeling. The need to focus on undergraduate immunology education has been strongly recognized and advocated for in several recent publications. In response to this need, we, a newly formed task force of undergraduate immunology educators, have identified fundamental concepts that are critical to the understanding of immunology for undergraduate students. We have aligned these fundamental statements with the overarching concepts described in the AAAS report “Vision and Change in Undergraduate Biology Education: A Call to Action”, and are now gathering feedback on these guidelines from undergraduate educators at large. Establishment of these guidelines is consistent with new pedagogical practices that emphasize conceptual understanding of the subject over rote memorization and provides a framework needed to introduce students to immunology. The goal of this project is to solicit feedback from the immunology community in an effort to achieve consensus regarding the fundamental concepts in immunology that are critical for STEM-focused students to comprehend.
Improving the computational reproducibility of immunological research
Authors: Anderson Brooke , Lyons Michael , Henao-Tamayo Marcela I , Juarrero Mercedes Gonzalez , Robertson Gregory
Published: January 2023

Many excellent and free training resources exist to improve the computational reproducibility of biomedical research. However, most of these materials target researchers at the stage of data analysis, and they typically provide much less guidance on principles and techniques to improve the reproducibility of the earlier steps of experimental data recording and experimental data pre-processing that are critical for ensuring the reproducibility of immunological research. Many common existing practices---including the use of spreadsheets with embedded formulas that concurrently record and analyze experimental data, problematic management of project files, and reliance on proprietary, vendor-supplied point-and-click software for data pre-processing---interfere with the transparency, reproducibility, and efficiency of laboratory-based immunological research projects. Under an NIH grant, we are creating new training modules as a resource for laboratory-based biomedical researchers to learn how simple computational principles can improve reproducibility at the stages of data recording and pre-processing in immunological research. In this poster, we will highlight several simple approaches that we have identified through this project to improve the reproducibility of immunological research, including strategies for re-designing data collection spreadsheet templates to more easily integrate into a reproducible data analysis workflow and the use of reproducible reporting tools within a laboratory setting.
Use of crossword puzzles: Filling the rows and columns to promote student engagement in an Immunology class
Authors: Kucknoor Ashwini S
Published: January 2023

Although crossword puzzles have been around for decades, researchers still study their use in the classroom. Previously, educational crossword puzzles only came in mass-produced student workbooks, which meant educators were unable to adapt them for the needs of their classroom. Although the research for teaching with educational crossword puzzles covers all sorts of subjects from ESL to Chemistry and widely used in grade schools mainly as a tool to learn vocabulary, studies find that crossword puzzles can help introduce and reinforce concepts for any subject. The bulk of undergraduate teaching is usually done through traditional PowerPoint lecture presentations in majority of the universities, which fails to encourage knowledge retention and learning. In contrast, recent studies have shown that the implementation of active- and collaborative learning, using games, music and puzzles promote stimulating environment to enhance student engagement and active participation of students during lecture. Active learning exercises provide major benefits such as the development of critical thinking, communication skills, and increasing motivation to learn. To this end, I have incorporated the use of crossword puzzles in my Microbiology and Immunology courses recently. I have developed questions covering new vocabulary or technical terminology, ability to distinguish between similar terms, evaluating options, and developing logical thinking for both Immunology and Microbiology concepts. I have mainly used the crossword puzzles to quickly summarize and emphasize the important facts and concepts delivered during regular lectures. The details of how this was used, and more ways by which this can be adopted in a course will be discussed.
METABOLIC INSULTS ELICIT EPITOPE-DEPENDENT IMMUNE AUTOREACTIVITY AGGRAVATING HEPATIC DAMAGE IN TYPE 2 DIABETES
Authors: Santambrogio Laura , Clement Cristina C , Nanaware Padma , Stern Lawrence
Published: January 2023

A diet rich in saturated fat and carbohydrates causes low-grade chronic inflammation in several organs including the liver, ultimately driving non-alcoholic steatohepatitis. The environment-driven lipotoxicity and glucotoxicity induces liver damage, which promotes dendritic cell adjuvanticity and generates an MHC-II immunopeptidome enriched of peptides derived from proteins involved in cellular metabolism, oxidative phosphorylation, and stress responses. Here, we demonstrate that metabolic insults promote the presentation of nested T and B cell epitopes from protein disulfide isomerase family A member 3 (PDIA3, which is involved in immunogenic cell death) on MHC Class II, ultimately supporting pathogenic autoreactivity. Additionally, PDIA3-driven T cell responses infiltrating the hepatic parenchyma, favored the release of TH1 and TH17 pro-inflammatory cytokines and, PDIA3-driven B cell responses were associated with antibodies that aggravated hepatic toxicity in mice subjected to high-fat and high-sucrose diet. Increased humoral responses to PDIA3 were also observed in patients with chronic inflammatory liver conditions including autoimmune hepatitis, primary biliary cholangitis and type 2 diabetes. Altogether our data indicate that metabolic insult caused by high-sucrose diet, cause tissue damage, which, by supplying PDIA3 T cell and B cell epitopes further contributes to epitope-dependent pathogenic immune autoreactivity

Supported by NIHAI146180 and NIH AI137198
Sequence similarity between SARS-CoV-2 Nucleocapsid and CNS proteins provides mechanistic insight into viral neuropathogenesis following infection
Authors: Lake Camille , Breen Joseph
Published: January 2023

The novel coronavirus SARS-CoV-2 emerged in 2019 and continues to cause death and disease throughout the world. To date, there have been 272 million infections and 5.3 million deaths worldwide, underscoring the necessity of understanding the virus and host immune response as well as developing novel therapeutics for severe disease. From the start of the pandemic, a prominent pattern of central nervous system (CNS) pathologies – including cases similar to Multiple Sclerosis (MS) - has emerged that suggest a related, underlying mechanism of viral mimicry to proteins expressed by CNS tissues. We hypothesized that immunodominant epitopes of SARS-CoV-2 share homology with proteins typically associated with MS. Using Pepmatch, a bioinformatics package which predicts peptide similarity in the context of MHC presentation, we discovered that Nucleocapsid protein, but not Spike or Membrane proteins, of SARS-CoV-2 shares significant overlap with 22 MS-associated proteins. To provide further physiological relevance, we used an MHC binding prediction tool to determine whether these Nucleocapsid peptides would bind preferentially to MS-associated alleles. While we did not find any significant binding pattern among MS-associated alleles, we did discover that Nucleocapsid peptides which overlap with CD99 Antigen were among the top HLA-peptide predictions. This finding provides a plausible mechanism by which the targeting of CD99 by lymphocytes leads to compromised blood-brain-barrier integrity and exacerbated viral neuropathogenesis. This work provides a deeper understanding of the unique viral dynamics with the human immune system and corroborates the targeting of CD99 to ameliorate neurological complications of SARS-CoV-2.
Molecular chaperone GP96 is a potential target to modulate dendritic cell programming and shape anti-tumor immunity
Authors: Mazzoccoli Luciano , Iwanowycz Stephen , Peterson Chelsea , Ngoi Soo , Hill Megan , Liu Bei
Published: January 2023

Dendritic cells (DCs) are professional antigen-presenting cells providing costimulatory signals to adaptive immune cells. Currently, the lack of strategies to improve antigen presentation is a hindrance in tumor immunology. Despite studies focusing on antigen cross-presenting Type-1 DCs (DC1s), a study published by our lab revealed Type-2 DCs (DC2s) with significant survival across multiple human cancers. Our research group has a long-term interest in studying immune chaperone GP96, which is an essential molecular chaperone for TLRs, GARP, and other vital innate receptors. The immune-related client network of GP96 creates opportunities to unmask the roles of multiple receptors on DCs. We have generated DC-specific GP96 deficient mice and reported increased tumor-infiltrating DC2 and delayed tumor development on the spontaneous breast cancer model. However, the mechanism by which GP96 regulates DCs function is under investigation. Using different tumor models, we found that deletion of GP96 on DCs improved immune response and decreased tumor growth. However, macrophage-GP96KO mice showed a loss of benefits observed on DC-GP96KO mice. Also, differential stimulatory/inhibitory molecules on DC1 vs. DC2 were tumor-dependent. To determine the role of DC-intrinsic GP96 in T cell activation, we used in vitro antigen-cross presentation assay. We found that OT-I CD8+ T cell activation was delayed at the initial days of the co-culture with GP96KO DCs but restored in the late phase compared with WT DCs. Collectively, the results show beneficial inflammatory DC2 molecular activation, with DC1 able to cross-present antigens to CD8+ T cells. Our study indicated that targeting GP96 on DCs may contribute to shaping T cell anti-tumor immunity.

Supported by grants from NIH NCI (R01:CA193939) and NIH NIAID (U01:AI125859)
B cell subsets contributing to the autoreactive plasma cell pool in Lyn−/− mice
Authors: Satterthwaite Anne B , Schneider Jalyn , Ottens Kristina
Published: January 2023

Systemic Lupus Erythematosus (SLE) is characterized by autoantibodies (autoAbs) against nucleic acid containing antigens. These antibodies form immune complexes that promote inflammation and cause organ damage. Defining B cell subsets that give rise to these autoAbs may reveal therapeutic approaches for SLE that spare responses to foreign antigens. Mice lacking the tyrosine kinase Lyn, which limits B and myeloid cell activation, develop lupus-like autoimmune disease characterized by a large increase in autoreactive plasma cells (PCs). We used several strategies to identify the origin of these PCs. We used fate mapping to assess the contribution of T-bet+ age associated B cells (ABCs), a subset that is increased and thought to be pathogenic in both murine and human lupus. 40% of splenic PCs in Lyn−/− mice were derived from T-bet expressing cells, a significant increase compared to wild type mice that was not seen in the bone marrow PC population. Marginal zone (MZ) B cells, also implicated in some lupus models, are enriched in autoreactivity, prone to PC differentiation, and reduced in Lyn−/− mice. We hypothesized that this reduction might be due to their unchecked transit to the PC stage. However, impairing MZ B cell development in Lyn−/− mice did not reduce autoAbs. Nor did preventing PC differentiation restore MZ B cell numbers. Instead, Lyn−/− mice accumulated B1a cells when PC differentiation was impaired. B1a cells tend to be polyreactive or weakly autoreactive and are primed for terminal differentiation. Our results suggest that both ABCs and B1a cells, but not MZ B cells, contribute to the autoreactive PC pool in Lyn−/− mice. Future studies will assess the role of these subsets in the development of autoimmune disease.

Supported by grants from the NIH (R21 AI161307, R21 AI137746)
NADPH oxidase-derived superoxide promote autoreactive T cell infiltration into islets of prediabetic NOD mice.
Authors: Blum Samuel I , Barra Jessie , Baker Rocky , Tse Hubert Ming
Published: January 2023

Proinflammatory cytokines, reactive oxygen species (ROS), and T cells contribute to β-cell destruction in Type 1 diabetes (T1D). NADPH oxidase (NOX) generate superoxide, a precursor for a variety of ROS, regulate cell survival, differentiation, signaling, and autoimmune responses in T1D. Previously, we demonstrated that NOX-derived superoxide increase chemokine synthesis and skew macrophages and T cells towards an inflammatory response in T1D. However, it is unclear if NOX is required for diabetogenic T cell trafficking to islets. Using non-obese diabetic (NOD) mice deficient in NOX-derived superoxide (NOD.Ncf1m1J), we tested the hypothesis that loss of NOX-derived superoxide delays T1D by impairing autoreactive T infiltration of islets. Female NOD. Ncf1m1J mice (n=20) exhibit a significant delay in spontaneous T1D, as only 20% become diabetic by 40-weeks of age compared to 90% NOD mice (n=23). Immunophenotyping of 12-week-old NOD.Ncf1m1J mice revealed fewer pancreatic CCR2+ CD4+ (1.9-fold) and CCR5+ CD4+ T cells (2.1-fold) compared to NOD. NOD.Ncf1m1J islets also had reduced total CD4+ (1.8-fold), CD25+ CD4+(2.0-fold), and effector CD4+ T cells (2.2-fold) compared to NOD. Using tetramer staining to detect autoreactive T cells, we observed a decrease in NOD.Ncf1m1J islet-infiltrating CD4+ T cells specific for insulin (Ins) (2.6-fold), Ins-chromogranin A hybrid peptide (HIP) (3.0-fold), and Ins-islet amyloid polypeptide HIP (2.6-fold) compared to NOD. Our data provides evidence that loss of NOX-derived superoxide can impair autoreactive T cell activation and islet-infiltration as a potential mechanism to delay T1D. Future studies will determine if NOX regulates islet chemokine synthesis to induce T cell migration.

Supported by R01 DK126456 R01 DK127497 T32 GM109780 T32 AI007051 F31 DK130551
Regulation of T cell CD5 levels by BTLA
Authors: Adegoke Adeolu , Šedý John R , Ware Carl F , Baldwin Troy A , Anderson Colin C
Published: January 2023

Many coinhibitory receptors are absent from naïve T cells and upregulated upon activation. In contrast, there are a small number of coinhibitory receptors expressed constitutively by naïve T cells, including CD5, BTLA, and VISTA. The relationship between these constitutively expressed coinhibitors is unknown. We examined the relationship between the constitutively expressed BTLA and CD5 in T cell ontogeny. We found an inverse relationship between CD5 and BTLA expression levels, with low BTLA expression in the thymus and higher BTLA expression in the periphery, corresponding with high and low CD5 expression, respectively. To determine if there is a causal relationship between BTLA expression and CD5 expression, we examined CD5 expression in wild type (WT) vs. btla−/− T cells. Interestingly, btla−/− T cells consistently expressed higher levels of CD5 both in thymic and splenic CD4 and CD8 T cells, indicating that BTLA expression directly or indirectly determines the level of CD5 expression broadly across T cells. In contrast, the loss of an inducible coinhibitor, PD-1, only affected CD5 levels on splenic CD8+ T cells. The loss of BTLA expression early in ontogeny might alter the TCR repertoire indirectly affecting CD5 levels, or instead its loss might affect CD5 expression rapidly within mature T cells. To examine the latter, we deleted btla by administering tamoxifen to adult btlafl/fl CreERT2+/− mice and WT CreERT2+/− controls. Loss of btla in adults led only to a transiently heightened CD5 expression. Together our data indicates that loss of BTLA early, but not later in ontogeny, leads to a long-term increase in CD5 expression by T cells. Such calibration of CD5 levels early in ontogeny might serve to reduce autoimmunity.

Funded by the CIHR, NSERC, and an AAI fellowship; btlafl/fl mice provided by the La Jolla Institute for Immunology.
Identification of the brain network controlling systemic inflammatory signals
Authors: Hashimoto Okito , Lee Diana , Hepler Tyler , Tsaava Tea , Tynan Aisling , Tracey Kevin J , Chavan Sangeeta S
Published: January 2023

Objective

Systemic inflammatory conditions are finely controlled not only by the immune system but also by the neuronal system in the body. While the vagus nerve is the key neuronal pathway to control the peripheral immune system, known as the inflammatory reflex, it is largely unknown how the brain regulates the reflex. The purpose of this study is to clarify the brain network response to systemic inflammatory signals.

Methods

Transgenic targeted-recombination-in-active-populations (TRAP2) mice that expressed tamoxifen-inducible Cre under control of an activity-dependent c-Fos promoter were crossed with a Cre-dependent tdTomato reporter line. These mice (TRAP2/tdTomato mice) were injected with 4-OHT (an active form of Tamoxifen) to induce Cre recombination and tdTomato expression. 30 min later, TNF was administered intraperitoneally to these mice and the brains were collected 7 days after the induction.

Results

Nucleus of the solitary tract (NTS), where vagal afferents ascending from peripheral tissues synapse on neurons, showed the increased number of tdTomato-expressing cells. In addition, TNF stimulation reinforced the tdTomato expression in the paraventricular nucleus (PVN) and the bed nuclei of the stria terminalis (BNST). Interestingly, many tdTomato-expressing cells colocalized with the sub-nuclei of PKC delta-positive neurons within the BNST.

Conclusions

Systemic TNF signals are transmitted to the brain regions through the vagus nerve, including NTS, PVN, and BNST. Furthermore, the specific neuronal population within the BNST may play a role in the modulation of systemic inflammatory condition.

This experiment is supported by grant from NIH to KJT and SSC

Supported by grants from NIH (R01GM132672, R35GM118182-01)
Using Team Based Learning to Solidify Immunology Concepts
Authors: Turula Holly , Bauler Timothy
Published: January 2023

Using Team-Based Learning (TBL) in the classroom allows learners to synthesize complex information to solve real-world problems. Through TBL learners distill the most salient points and draw them together in a way that fosters higher order thinking. Similarly to immune cells, we work most effectively in collaboration. During TBL learners simulate complex situations and participate in peer teaching and learning. In these ways TBL fosters mentorship and a sense of community. Not only that, but learners enjoy it and find it beneficial! Learning how to implement TBL in the classroom to help engage the learners benefits not only the student, but the professor, the institution, and the field of Immunology.
Instagram-a non-conventional learning tool in post-covid educational world. Repurposing of an entertainment app.
Authors: Fazal Nadeem , Brehm Danielle , Alakbarova Gulgaz , Huseynbayli Ayshan , Garajayev Azer , Safarova Arzu , Cebiyev Nihat , Meher Satish , De Herde Laura
Published: January 2023

Who would have thought that social media will ever be employed as a non-conventional learning tool. Before March 2020 everybody would have raised eyebrows and shunned the practice of even going in that direction. Pandemic changed the world! A tool of pure entertainment and pleasure had a positive effect on the generations “Z” and “X”. Little did the educators realize that one of the most intrinsic tool of self-directed learning is to stay “relevant” stay “motivated: and staying “alive” in a COVID-19 deadly environment was an app called “Instagram”. We conducted a global survey from university students across Asia, Europe, Russia, and Americas. In the current study we used “instagram” as a tool to collect anonymous data from more than 500 respondents and summarized the following points. Firstly, 80% of the respondents were females, and between the age group of 15–25 years. Only 9% reported negative mental health issues while the rest of respondents reported a positive, competitive, and highly focussed group of students who helped each other in small or large social circles to keep reminding each other of educational needs, helping with difficult assignments, and small group studying and quizzing each other. 50% of the respondents reported to get more work accomplished by communicating through messaging, pictures and videos. There was a small 10% of the students who got distracted and sometimes would get carried away but still performed well in learning objectives as compared to those who did not join the trends. 30% of the students did notice impulsiveness, increased anxiety and burnt out by excessive multitasking. Overall, we report that the best purpose for the use of instagram may not be a tool in teachers locker but definitely a peer-mentoring source.