bluuebunny/crossref_metadata_2025_split huggingface dataset:
Insulin specific TCR repertoire analysis reveals functional diversity of Treg TCRs
Authors: Jing Yi , Kong Yuelin , Trout Denise , Bettini Matthew L , Bettini Maria 
Published: January 2023

Abstract
Regulatory T cells (Tregs) are critical in preventing autoimmunity and express a unique TCR repertoire with high affinity to self-antigens. However, whether Treg and effector T cell (Teff) TCR repertoires are similarly divergent in the context of autoimmunity, and whether Tregs are able to maintain TCR affinity advantage has not been clearly defined. Insulin tetramer positive Tregs isolated from pancreatic islets of NOD mice showed a higher level of self-reactivity (CD5) and TCR signaling (Nur77-GFP) than Teffs specific to the same epitope. We isolated and sequenced insulin tetramer+ Teffs and Tregs from islets of NOD mice expressing a fixed TCRα of an insulin specific TCR. Surprisingly, significant overlap was identified between Treg and Teff TCR repertoires, suggesting that similar TCRs can support autoimmune Treg and Teff development and recruitment into the autoimmune response. Both Tregs and Teffs exhibited TCRs with a broad range of reactivity to insulin epitope and Treg TCRs did not show increased reactivity. The disconnect between Nur77-GFP and TCR functional affinity suggests a Treg cell-intrinsic capacity to compete for antigen or to enhance downstream signaling. When re-expressed in vivo using the TCR retrogenic method, Treg derived TCRs showed lower Treg generation and collectively higher diabetogenicity. With comparable Treg generation, low affinity Treg TCR showed significantly lower protection against diabetes than high affinity Treg TCR. These findings indicate that in tissue specific autoimmunity Tregs can express a TCR repertoire with a broad range of functionality and have significant overlap with Teffs of the same specificity, which might negatively affect their suppressive capacity.
This work was supported by the NIH (AI125301-01A1) and The Robert and Janice McNair Foundation.
Profiling transcriptomes, TCR repertoires, and antigenic specificities of islet-infiltrating T cells in Non-Obese Diabetic mice
Authors: Zdinak Paul , Grebinoski Stephanie J , Torrey Jessica , Rathod Sanjay , Ranjan Rashi , Hicks Louise , Joglekar Alok V 
Published: January 2023

Abstract
Type 1 Diabetes (T1D) is an autoimmune disease caused by progressive destruction of pancreatic β-cells, resulting in reduced insulin production. The role of islet-infiltrating CD4+ T cells in mediating β-cell destruction and autoantibody formation is well established. However, many of the antigen specificities of CD4+ T cells which initiate disease are undefined. In this project, we performed single cell RNA sequencing of pancreatic islet infiltrating T cells from 6-, 8-, and 10-week-old Non-Obese Diabetic (NOD) mice. NOD mice recapitulate many immunologic features of T1D and share many autoantigens with human T1D patients. Analysis of TCR repertoires revealed clonal expansion in activated and pre-exhausted effector CD4+ T cells as well as regulatory T cells. Despite subtle differences, expanded effector CD4+ T cells showed a well-defined cell state. We cloned 40 TCRs from the top expanded T cells and expressed them in Jurkat cells. We performed antigen discovery assays on these TCRs using our lab’s Signaling and Antigen-presenting Bifunctional Receptor (SABR) platform. We constructed a SABR library of 4,075 I-Ag7-restricted epitopes based on published datasets. We identified several previously reported and novel autoantigens. Surprisingly, antigenic specificities did not influence the shared phenotype dramatically. These results identify several potential autoantigens that will be valuable for diagnostic, preventative, and therapeutic purposes. Moreover, these results suggest that the islet microenvironment is a major determinant of infiltrating CD4+ T cell states. Similar studies focused on CD8+ T cells in NOD mice and CD4+ and CD8+ T cells in humans are underway.
dkNET New investigator Pilot Program in Bioinformatics (PI: Joglekar) NIDDK New Investigator Gateway Award 1R03DK127447 (PI: Joglekar) PACER Innovative Discovery Award (PI: Joglekar)
Control of medullary thymic epithelial cell development and central tolerance by a zinc finger transcription factor
Authors: Sin Jun Hyung , Costa Juliana Sucharov , Waterfield Michael 
Published: January 2023

Abstract
We have identified a novel function for a zinc finger transcription factor in medullary thymic epithelial cell (mTEC) development and central tolerance. Previous studies have shown that the transcription factor has critical functions in immune cell development, however our study is one of the first to describe a role for the transcription factor in the epithelial lineage. Probing publicly available gene expression databases, we found that the transcription factor is highly expressed in mTECs. Recent studies on mTECs have shown considerable cellular heterogeneity within mTECs with at least five mTEC subtypes including a transient amplifying population (TAC-TECs), autoimmune regulator (Aire+) mTECs, Late/Post Aire mTECs, Ccl21a mTECS, and tuft cells. Using a conditional knockout mouse line, we deleted the transcription factor in mTECs which resulted in drastic changes in the cellular composition of mTECs with substantial increase in Ccl21a mTECs and tuft cells and a dramatic decrease in Aire+ mTECs and Late Aire mTECs. Furthermore, the remaining Aire+ mTECs have a significant decrease in tissue-specific antigen gene expression resulting in autoimmunity in mice. To gain mechanistic understanding of the developmental changes we observed, we performed scRNA-seq and scATAC-seq, which showed significant changes in gene expression and the chromatin landscape in mTECs and helps to explain the critical function for this transcription factor in mTEC development.
The influence of antigen recognition on Treg suppressive function
Authors: Rase Viva Jeanne , Kolawole Elizabeth M , Evavold Brian D 
Published: January 2023

Abstract
Tregs play an essential role in preventing autoimmunity by maintaining effector T cell balance and self-tolerance. Antigen stimulation through T cell receptor (TCR) makes Treg suppression more efficacious and as such is generally viewed as a strong predictor of Treg function. In demyelinating disease Tregs with the most effective suppressive capacity are self-antigen specific, however, it is unclear how the parameters of self-antigen stimulation (affinity, force, and bond lifetime) modulate Treg suppressive potency. Here we utilize the well-described myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide driven experimental autoimmune encephalomyelitis (EAE) to address how Treg self-antigen interactions under chronic inflammation influence suppressive potency. Recently Tregs have been shown to perform trogocytosis to remove MHC molecules from the surface of antigen presenting cells in an antigen specific manner. We hypothesize that trogocytosis correlates with optimal Treg antigen specific suppression in demyelinating disease. To test suppressive capacity of self-antigen specific Tregs we utilized a MOG knockout mouse model of EAE. In this system we show that MOG knockout Tregs are less potent suppressors when challenged with EAE despite identical affinity for MOG35–55 as wild type Tregs. In addition, we show that MOG knockout Tregs perform trogocytosis less readily than wild type Tregs demonstrating that the presence of self-antigen MOG35–55 is necessary for development of optimal Treg generation. An understanding of how Tregs respond to antigen may allow the design of therapeutic strategies to better harness Treg power in combatting autoimmune conditions.
Supported by grants from NIH (5R01NS071518-09)
PD-1 licenses activated Treg for lymphatic migration.
Authors: Piao Wenji , Li Lushen , Saxena Vikas , Hippen Keli , Bruce Blazar , Riella Leonardo , Bromberg Jonathan 
Published: January 2023

Abstract
Rather than serving as fluid transport conduits, the lymphatic vessels have been proved to be critical portals for immune cell migration. As non-immune cell, the lymphatic endothelial cell (LEC) constitutively express high level of programmed death-1 (PD-1) ligand (PD-L1), known as an immune checkpoint protein. We hypothesized that LECs use PD-L1 to regulate PD-1-expressing immune cells during transendothelial migration (TEM). Activated T cells expressed substantially elevated levels of both PD-1 and PD-L1. For human and mouse Tregs PD-1 was in excess of PD-L1, while effector CD4 T cells (Teffs) expressed PD-L1 in excess of PD-1. PD-1−/− but not PD-L1−/− Tregs had reduced migration and motility compared to WT. Engagement by PD-L1 Fc induced rapid activation of Akt but suppressed classical NFkB-P65 signaling in Tregs. WT LECs constitutively expressed high level of surface PD-L1. PD-L1−/− LEC or lymphatic vessels (LV) had increased adhesion and junction proteins VCAM-1 and zonulin-1, and had more zipper junctions. PD-1 triggered PD-L1 signaling in LEC result in strong PI3K/Akt and NFkB-p65 responses that regulated endothelial structure and enhanced TEM. Similar changes to LEC junctions were observed when PD-1high Tregs migrated across LEC layers, resulting in rapidly induced NFkB-p65 translocation into the LEC nucleus. PD-1-induced PD-L1 signaling decreased VE-cadherin expression, which was restored by blocking ERK and PI3K/Akt. Importantly, PD-1-blockade of intra-tumoral Tregs reduced tumor egress and the detained Tregs converted to IFN-g-producing Th1Tregs. These data demonstrate novel roles for Treg PD-1 and LEC PD-L1 in regulation of lymphatic migration and ultimately Treg suppressive function.
Supported by grants from NIH (2R37AI062765-17A1)
cGAS-STING Mediated Inflammation In Diabetic Wound Macrophages Is Regulated By Histone Demethylase JMJD3
Authors: Audu Christopher O , Melvin William J , Wolf Sonya , Sharma Sriganesh B , Mangum Kevin D , Barrett Emily , Joshi Amrita , Gallagher Katherine 
Published: January 2023

Abstract
Recent work has shown that the cGAS-STING inflammatory pathway is elevated in conditions that are marked by chronic inflammation, such as obesity and diabetes. Using single cell RNA (scRNA) sequencing from human wounds, we show that the Sting gene is increased in diabetic wound macrophages. Histone demethylase JMJD3 specifically demethylates lysine 27 on histone 3 (H3K27me3) - a repressive mark for gene transcription. Using diabetic mice, we note on ChIP analysis, a decrease in the H3K27me3 repressive mark at the STING promoter in diabetic wound macrophages relative to littermate controls, with subsequently enhanced STING protein levels by western blot. Additionally, scRNA from wound macrophages obtained from a novel murine strain deficient in Jmjd3 in myeloid-cell lineages (Jmjd3f/f Lyz2Cre+), revealed significantly decreased Sting gene levels. Further, ChIP analysis of H3K27me3 levels at the STING promoter from Jmjd3f/f Lyz2Cre+ wound macrophages reveal increased H3K27me3 levels compared to littermate controls, with a concomitant decrease in STING protein as determined by western blot. Mechanistically, we use mRNA, protein and ChIP studies to reveal that in diabetic wound macrophages, IL6 is responsible for increased JMJD3 leading to increased JAK1,3/STAT3 activity and increased STING production. Wound healing curves from diabetic mice locally treated with macrophage-specific nanoparticles against JMJD3 showed significantly improved healing, and decreased STING protein production in these wounds. Taken together, this data highlights a unique epigenetic regulator of STING production in diabetic wound macrophages and provides a viable cell-specific, nanotherapeutic approach to non-healing diabetic wounds.
Supported by NIH (F32DK126471), Vascular and Endovascular Surgical Society Resident Research Award
Elucidating the mechanism of neutrophil-mediated lung injury in sepsis and the role of CD11d/CD18 integrin on this process
Authors: Koutsogiannaki Sophia , Yuki Koichi 
Published: January 2023

Abstract
Sepsis is the leading cause of death in intensive care unit (ICU) and the most expensive condition treated in the US, without a specific therapy yet available. Acute lung injury (ALI) is one of the most significant organ injuries in sepsis, resulting from massive migration of neutrophils to the lung, but the exact mechanism is not known. β2 (CD18) integrin family [consisting of αLβ2 (CD11a/CD18), αMβ2 (CD11b/CD18), αXβ2 (CD11c/CD18) and αDβ2 (CD11d/CD18)] and its counter-receptor on the endothelium, ICAM-1 (CD54), are critical for neutrophil migration, but their role in ALI is not yet delineated. Using a murine model of sepsis induced by cecal ligation and puncture (CLP) surgery, we showed decreased neutrophil levels and attenuated injury in the lung of β2−/− and ICAM-1−/− mice at 12h post-CLP, suggesting the importance of β2 integrins in this process. In addition, we observed decreased neutrophil levels and attenuated injury in the lung of αDβ2−/− mice but not in the lung of αLβ2−/− and αMβ2−/− mice, suggesting that αDβ2 has an orchestrating role in sepsis-induced ALI. In support, we found increased αDβ2 expression levels on neutrophils both in the lung and blood of WT mice as sepsis progressed. RNAseq analysis in neutrophils from blood and lung of WT and αDβ2−/− mice showed that αDβ2 mediates neutrophil migration to the lung through pathways associated with antigen presentation, apoptosis and NOD-like receptor signaling. αDβ2−/− neutrophils had also less Cxcr2, Ltb4r1 and Dhrs9 expression, associated with neutrophil migration and the retinoic acid pathway. Taken together, our results suggest a mechanism for ALI in sepsis in which αDβ2 integrin has a major role and could be a novel target for therapeutic intervention.
The Anesthesia Research Distinguished Trailblazer Award, Boston Children’s Hospital The William F. Milton Fund, Harvard University
STK25 acts as a regulator of leukocyte migration
Authors: Rice Matthew R , Matta Bharati , Barnes Betsy J 
Published: January 2023

Abstract
The recruitment of leukocytes to sites of infection is an essential component of the innate immune response to pathogens. Defects in leukocyte migration can render the host susceptible to recurrent infections. In our efforts to identify new mediators of innate immunity, we found that serine/threonine protein kinase 25 (STK25) promotes TLR-induced proinflammatory cytokine production. While STK25 has previously been implicated in the regulation of cell migration, its role in leukocyte trafficking has not been defined. To investigate the involvement of STK25 in leukocyte migration, we evaluated baseline hematological parameters from Stk25-deficient (Stk25−/−) mice via a Hemavet. We found that Stk25−/− mice exhibit reduced circulating lymphocytes, neutrophils, and monocytes at steady-state compared to age- and gender-matched wild-type littermate (WT) mice. Immunophenotyping by flow cytometry confirmed these findings and revealed a significant reduction in the frequency of Ly6Chi inflammatory monocytes in Stk25−/− peripheral blood (PB) relative to WT. In contrast, we observed no apparent change in the frequencies of neutrophils and Ly6Chi inflammatory monocytes in Stk25−/− bone marrow, suggesting a defect in leukocyte migration. Finally, we examined the role of STK25 in neutrophil trafficking via an in vitro chemotaxis assay. We found that PB neutrophils from Stk25−/− mice displayed a defect in migration toward WT serum, suggesting a cell-intrinsic role for STK25 in the control of cell trafficking. Additionally, PB neutrophils from WT mice exhibited reduced migration toward Stk25−/− serum, supporting a cell-extrinsic function for STK25. Thus, our data support a new role for STK25 as a mediator of leukocyte trafficking.
Mechanical stress modulates NLRP3 pathway in macrophages
Authors: Joshi Hemant , Almgren-Bell Alison , Todd Elizabeth M , Morley Sharon Celeste 
Published: January 2023

Abstract
NLRP3 inflammasome activation releases the pro-inflammatory cytokine IL-1b and induces pyroptotic cell death. Dysregulated NLRP3 activation escalates inflammatory diseases such as acute lung injury (ALI), ventilator-induced lung injury (VILI), liver fibrosis, and idiopathic pulmonary fibrosis (IPF). These inflammatory disorders are associated with increased mechanical stress due to changing tissue compliance/stiffness. Resident macrophages sense this mechanical signal via a process known as “mechanotransduction”. However, the underlying mechanism of how mechanotransduction regulates the NLRP3 inflammasome is not fully known.
In this study, we are investigating that how NLRP3 pathway is regulated in lung resident macrophages during mechanical stress. To alter substrate compliance, macrophages are cultured on collagen-coated gels with various physiologically relevant stiffness (elastic moduli; measured in kiloPascal (kPa)) prior to NLRP3 activation. We have found that the NLRP3 inflammasome is sensitive to the substrate stiffness. NLRP3 induced IL-1β and pyroptosis-inducing gasdermin-D production were highest on the softest substrate (0.2 kPa) and decreased on a stiffer substrate (64 kPa). However, NLRC4 inflammasome activation was not mechano-responsive, suggesting ASC adaptor mediated cross signaling in mechanotransduction. Further analysis of gene expression using high-throughput RNA-sequencing, revealed altered expression of inflammatory bio-markers on macrophages when substrate stiffness was varied. Further examination of molecular interactions between adhesion-based mechanotransduction and NLRP3 signaling will help in understanding inflammatory diseases such as lung fibrosis.
AAI career in Immunology Fellowship, R01-AI104732 and R56 AI104732
Incorporating Immunology into the Undergraduate Curriculum to Promote Interdisciplinary Science Education
Authors: Pandey Sumali , Justement Louis B , Taylor Rebekah 
Published: January 2023

Abstract
The national call for undergraduate education reform in the Vision and Change report identified understanding the importance of interdisciplinary science as a key competency; one that is critical for career success and responding to the evolving needs of the STEM workforce. Immunology provides an ideal platform to foster an appreciation of the interrelationships between multiple disciplines in the natural sciences. Interdisciplinary thinking can be fostered by creating educational interventions that relate concepts in immunology with foundational concepts in other sciences and is critical to tackle current societal challenges, such as the effect of global climate change on emerging infectious diseases and the need for new strategies to combat these threats. Although the discipline of immunology integrates easily with other biological fields such as microbiology and physiology, incorporating interdisciplinary thinking into broad educational practice requires intention and collaboration. In this breakout session, we will introduce a conceptual framework that: i) identifies interdisciplinary immunology focused learning outcomes; and ii) demonstrates classroom strategies to address these outcomes. Participants will engage in an active discussion to better understand ways in which Immunology education can be refocused to enhance interdisciplinary learning. The goal of the session is to empower educators by providing a conceptual framework that will enable them to mindfully and intentionally incorporate backward design into their pedagogy to teach interdisciplinary science using immunological topics.
Supported by grant from NSF (#2120806)
Driving social belonging and content retention by using the Flipgrid video discussion app.
Authors: Lukin Kara , Aviszus Katja , Nardi Chelsey 
Published: January 2023

Abstract
Even strong students struggle with the level of detail and interconnectedness required to master immunology. We incorporated Flipgrid assignments to leverage social and emotional learning and cognitive science to enhance students’ engagement with and understanding of content in an undergraduate-level immunology course. The tool is appropriate for elementary through professional school students. Flipgrip is a simple, video recording app used via a cell phone or computer. It can be integrated with most learning management systems. Students created short videos of themselves explaining learning objectives they selected. The videos were evaluated by the educator, updated if needed and shared with the class as tools for review. Peer-evaluation is possible, too. This feedback can be provided quickly in the app.
Through such activities, students increase their sense of social connectedness and belonging which research has shown enhances 1) intrinsic motivation and course engagement, 2) safety in the learning community so students ask peers and educators for support, and 3) overall academic success and retention. The benefits of belonging are especially beneficial for students from historically excluded and underrepresented groups. The approach allows students to become experts in a concept and contribute to discussions in ways they might not feel comfortable doing during class sessions. The videos provide opportunities to see and celebrate diversity among the class in a variety of ways. The creativity students bring to the videos help generate recall clues that allow students to retain information more effectively. Similarly, the collection of videos allows students to use distributive recall to self-assess and solidify content.
Supported by No funding support
Pharmacy Immunology-challenges of teaching a new course in a COVID-affected world of education.
Authors: Fazal Nadeem 
Published: January 2023

Abstract
Immunology education in US schools and colleges of pharmacy do not have a standardized curriculum. Half of 142 colleges of pharmacies have a dedicated course of Immunology with three fourth of them in the first year of pharmacy education. Although it is a part of core curriculum yet more private than public schools allocate about 3 credit hours. Chicago State University College of Pharmacy offered a new 2-credit course of “Pharmacy Immunology” in the fall of 2021 to twenty-four second year pharmacy students for the first time. Ten lectures on basic immunology, i.e., innate and adaptive immunity, prepared the students to understand disorders and diseases associated with the immune system. Four workshops of 3-hours each, on Hypersensitivities, Autoimmunity, Transplantation and Tumor Immunology were added to the syllabus. The workshops employed a Modified Flipped Classroom, Team-Based Learning (TBL), Case-Based Learning (CBL), YouTube videos as a combination of various teaching methods. The course was offered as a standard in-class on-campus module. Student’s performance was assessed by tRAT and iRAT. Data of student performance and perception was collected and analyzed. 58.3% of students agreed that the workshop’s mode of learning helped them. TBL appealed to 29.2%, CBL 45.8%, and PowerPoint lectures 79.2% of the students. Overall, 33.3% of the students disliked workshops as a tool for offering an interactive educational tool. Assessment data confirmed that the pass rate was 100%, with 20.8% passing in iRAT as compared to 79.2% in tRAT. Final assessment and course evaluation data suggest that a hybrid approach including a blended learning technique was the best option, catering for the needs of most students having a diverse learning style.