bluuebunny/crossref_metadata_2025_split huggingface dataset:
The histone methyltransferase Whsc1 regulates TGFβ-driven macrophage to myofibroblast transition
Authors: Mangum Kevin D , Joshi Amrita D , Melvin William J , Mazumder Purba , Erturk Asuman , Wolf Sonya , Barrett Emily C , Moon Jadie Yoonjoo , Obi Andrea , Kunkel Steven L , Moore Bethany B , Gallagher Katherine 
Published: August 2023

Abstract
In normal wound healing, macrophages regulate early and late inflammation, allowing for resolution of tissue injury and subsequent healing. During normal tissue repair, macrophages undergo transition to myofibroblasts, which promote wound healing. While this critical transition is impaired in diabetic wounds, the underlying mechanisms responsible are unknown. Our group and others have identified that epigenetic regulation of macrophages controls their phenotype in wounds. Since TGFβ drives macrophage to myofibroblast transition, we performed an epigenetic superarray to identify key chromatin modifying enzymes in macrophages (CD11b+CD3− CD19-NK1.1-Ly6G-) from normal diet (ND) and diabetic-induced obese (DIO) murine wounds treated ex vivo with TGFβ. The histone methyltransferase Whsc1, which mediates di- and tri-methylation at H3K36, was significantly increased in DIO macrophages with TGFβ treatment compared to ND macrophages (p&lt;0.05). siRNA knockdown of Whsc1 in BMDMs decreased TGFβ-dependent expression of fibrotic (Acta2, Col1a1, Col3a1) and inflammatory genes (IL-1b, IL-6, and IL-12) (p&lt;0.05). In ChIP experiments, Whsc1 was enriched at Col1a1 and Col3a1 promoters in ND but not DIO BMDMs, and TGFβ inhibited Whsc1 binding as well as H3K36me2 and H3K36me3 at fibrotic gene promoters. Additionally, H3K36me2 and H3K36me3 were decreased in Whsc1-knockdown BMDMs at Col1a1 and Col3a1, validating Whsc1 as an H3K36 methyltransferase that regulates myofibroblast gene expression. These results suggest that TGFβ regulates macrophage to myofibroblast conversion via Whsc1, which we identify as a critical epigenetic switch in tissue repair and potentially an important therapeutic target in diabetic wounds.
F32 DK131799-01, 2022 American College of Surgeons Resident Research Scholarship, 2022 VESS/Medtronic Resident Research Award
New insight into DC deficiencies in Chronic Lymphocytic Leukemia (CLL)
Authors: Welch Baustin M 
Published: August 2023

Abstract
Dendritic cells (DCs) function as principal sensors of PAMPs and DAMPs, connecting the innate and adaptive arms of the immune system. DC subset deficiencies have been observed in untreated CLL patients compared to age-matched healthy controls. Studies investigating the specific DC subsets that differ in function and localization; conventional DCs (cDCs), plasmacytoid DCs (pDCs), and Axl+ Siglec-6+ (AS) DCs are lacking in CLL. In mice, DC development and maintenance is highly dependent on the Flt3/Flt3L signaling axis. Activated T cells are believed to be the major source of Flt3L to DCs. Previous studies have reported elevated Flt3L in the serum of untreated CLL patients. Interestingly, increased serum levels of Flt3L have been associated with DC deficiencies, as DCs are believed to be the principle immune cell requiring this cytokine. This study will utilize extensive flow cytometry panels to investigate the prevalence and functional status of DCs in untreated CLL patient blood, including cDC1, cDC2, pDCs, and AS DCs. In addition, we will interrogate the ability of CLL-derived exhausted T cells to provide Flt3L upon activation.
A High Parameter Flow Cytometric Assay to Identify Human Myeloid Derived Suppressive Cells
Authors: Wang Xiaoyang , Anderson Keon , Chen Jingyi , Bonilla Diana , Deng Jun , Edinger Mark 
Published: August 2023

Abstract
The concept of myeloid derived suppressive cells (MDSCs) has emerged in recent years as a group of myeloid cells that pocess potent immunosuppressive activity. These cells expand under pathologic conditions and suppress the immune functions of T cells, dendritic cells, macrophages, and natural killer cells. They have been shown to be involved in the pathological states of cancer, autoimmune diseases, and infections, such as COVID-19.
MDSCs are subset into PMN (polymorphonuclear)- and M (monocytic)-MDSCs. Phenotypic markers of MDSCs have been elucidated in recent years. Here Cytek® developed a 14-color, 16-marker, single-tube flow cytometric assay to analyzed MDSCs. The panel includes markers to exclude lymphoid lineage cells, and markers to identify PMN-MDSC and M-MDSC populations, such as CD15, CD14, CD16, CD66b, CD11b and HLA-DR. In addition, PMN- and M-MDSC specific expression markers such as Lox-1, CXCR-1, DR5 are also included. Fresh whole blood samples were stained with erythrocyte lyse wash method and the samples were analyzed on a Cytek® Aurora or 3-laser Northern LightTM instrument. The panel performance was evaluated on blood samples collected in EDTA, Heparin, ACD, and Cytochex blood tubes as well as peripheral blood mononuclear cell (PBMC) samples. Assay precision was assessed by testing on 12 donor samples in triplicates on three consecutive days. Targeted cell populations are well identified in peripheral blood and PBMC samples with good resolution and assay precision with %CV less than 25% for all cell populations of interests.
This 14-color 16-marker preoptimized flow cytometric assay can be used in clinical research and drug discovery in cancer, autoimmune diseases, infections and other inflammatory diseases.
Pituitary immune cells regulate gonadotrope hormone secretion
Authors: Del Mundo Zena Marie , Nguyen Thu , Ujagar Naveena , Nicholas Dequina 
Published: August 2023

Abstract
Polycystic ovary syndrome (PCOS) is one of the most common causes of female infertility in the US and is associated with a combination of reproductive neuroendocrine physiological changes. Previous studies have shown that PCOS patients have elevated serum C reactive peptide and lipopolysaccharide (LPS), which indicates chronic low-grade inflammation. However, it is unknown how inflammation or immune cells impact reproductive outcomes. In this project, I study how inflammation affects gonadotrope regulation of sex hormone secretion. I show that in vitro depletion of CD45+ cells from dispersed primary mouse pituitary increases the luteinizing hormone (LH) to follicle-stimulating hormone (FSH) ratio. In contrast, immune activation through chronic LPS exposure in mice results in decreased serum LH to FSH ratio. Using single-cell RNA sequencing data from CD45+ enriched cells from mouse pituitaries, I identified specific resident immune cells. These cell populations include macrophages with higher RNA expression of heat shock proteins in male mice compared to female mice suggesting inflammatory protection in males. Ongoing experiments include chronic LPS exposure in mice, where pituitary cells are immunophenotyped, sex hormone serum levels measured, and ovulation monitored. Ultimately this work will allow us to understand the contribution of inflammation to PCOS, with the hope of designing immunotherapy for PCOS.
NICHD R00 HD098330-03
An immunosuppressive tumor population drives immunotherapy resistance of heterogeneous tumors
Authors: Tanaka Miho , Lum Lotus , Ledezma-Soto Cecilia , Hu Kenneth , Superville Daphne , Adams Zoe , Kersten Kelly , Krummel Matthew F , Reeves Melissa Q 
Published: August 2023

Abstract
Intratumoral heterogeneity (ITH) – cellular and molecular diversity within a tumor – is linked to failure of immunotherapy in multiple cancer types. A high degree of ITH is associated with poor infiltration of T cells into the tumor and resistance to immune checkpoint blockade (ICB) therapy. To determine how distinct tumor populations within heterogeneous tumors shape the immune microenvironment and how this impacts therapy response, we modeled heterogeneous tumors composed of an RFP-tagged immunosuppressive tumor population and a YFP-tagged pro-inflammatory tumor population. The resulting tumors contained a patchwork of distinct regions with YFP+ cells, RFP+ cells, or a mixture of YFP+ and RFP+ cells. Analysis of the immune infiltrates in each region revealed a higher frequency of total CD4 T cells, Th1 cells and IFNg+ CD8 T cells in YFP regions compared to RFP regions, whereas macrophages exhibited the opposite pattern. PD-1 blockade and CD40 agonist combinatorial antibody therapy induced an increase in Th1 abundance in RFP regions, but the treatment did not clear the tumors. Together, these results reveal that distinct regional immune infiltration pattens within the tumor are driven by the local tumor cells present in each region, and that the treatment-induced global improvement of Th1 infiltration alone is not sufficient to induce tumor clearance. Moreover, we identified Cx3cl1 as a driver of a dominant immunosuppressive tumor microenvironment, marked by an increase in immunosuppressive CD206+ macrophages and decrease in anti-tumoral neutrophils and monocytes. These results suggest that an immunosuppressive tumor population drives immunotherapy resistance of heterogeneous tumors and Cx3cl1 as a new therapeutic target.
Supported by grants from Parker Institute for Cancer Immunotherapy (Project Grant)
Enzymatic depletion of adenosine to overcome resistance to immune checkpoint inhibitors in non-small cell lung cancer
Authors: Sarkar Omar Sadi , Donninger Howard , Chew Lewis C , Eaton John , Yaddanapudi Kavitha 
Published: August 2023

Abstract
Despite the remarkable outcome of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC), a large number of patients remain unresponsive to this therapy. A key factor of the unresponsiveness to ICIs is intrinsic therapeutic resistance where monocytic myeloid-derived suppressor cells (M-MDSCs) contribute significantly via suppressing CD8+ T cell mediated anti-tumor response. Our results showed that in tumor microenvironment (TME), tumor cells derived PGE2; a prostaglandin that directly induces CD73 (an ecto-nucleotidase that converts AMP to immunosuppressive adenosine) expression in M-MDSCs; consequently, CD73 expressing M-MDSCs suppress T cells via adenosine thus confer resistance to ICIs. In addition, we have shown that the depletion of adenosine via PEGylated Adenosine Deaminase (PEG-ADA) can reinvigorate CD8+ T cells, ultimately enhance the anti-tumor immunity.
Targeting the intrinsic signaling of PD-L1 in human monocytes by a novel PD-L1 antibody to promote anti-tumor immunity
Authors: Hsu Michelle A , Liu Xin , Barham Whitney , Hirdler Jacob , Mao Zhiming , Lucien Fabrice , Dong Haidong 
Published: August 2023

Abstract
Although immune checkpoint blockade that targets the PD-L1 has revolutionized the treatment of human cancers, only a small proportion of patients positively respond to this form of treatment. A limitation of these therapies is that they only target and inhibit PD-L1’s extracellular function, which is to bind its receptor on PD-1 on T cells and inhibit T cell cytotoxicity. Recent discoveries have shown that PD-L1 expressed on tumor cells can signal intrinsically and independently of PD-1 ligation to promote tumor cell survival. Besides tumor cells, host myeloid cells have been shown to have high expression of PD-L1, therefore contributing to the immunosuppressive nature of the tumor. However, PD-L1 intrinsic signaling in human myeloid cells has not been studied. Therefore, the purpose of our work is to determine the intrinsic functions of PD-L1 in myeloid cells and how this impacts anti-tumor immunity. To study this, we used a novel PD-L1 antibody (H1A) that does not block PD-L1’s binding with PD-1, but directs PD-L1 for degradation, thereby decreasing total PD-L1 expression. Additionally, we developed a chimeric mouse model in which the murine PD-1/PD-L1 has been replaced with the human version. This allows us to evaluate PD-L1’s intrinsic function in vivo. In our preliminary studies, the degradation of PD-L1 in human monocytes led to increased chemokine release as well as increased conversion of effector T cells. Additionally, H1A treatment of tumor-bearing mice led to lower tumor burden in a CD8 T cell dependent manner. Based on these results, PD-L1 intrinsic signaling has the potential to regulate the immune-stimulatory response of monocytes, thereby limiting effector T cell responses against cancer.
Supported by grants from NIH (R0192389) and Mayo Clinic graduate school of biomedical sciences
Assessment of the effectiveness of team-based learning activities on learning outcomes in the undergraduate immunology classroom
Authors: Bruns Heather A , Baty Joshua 
Published: August 2023

Abstract
Immunology is inherently interdisciplinary. Understanding how the immune system functions requires knowledge from several scientific disciplines including molecular biology, cellular biology, genetics, and biochemistry. Furthermore, immunology is conceptually complex, requiring the identification of a plethora of immune components and mastery of a large volume of new vocabulary. These attributes can pose challenges to student learning in the undergraduate immunology classroom. Team-based learning (TBL) is a pedagogical method used to increase student engagement in learning, improve student collaboration, and develop communication skills. In a variety of educational settings, TBL activities have been shown to foster a deeper understanding of complex topics, increase student confidence in course content, and improve learning outcomes. In this study, we examined differences in the impact of traditional lecture versus TBL activities on student learning outcomes for four different topics presented in an undergraduate adaptive immunity course. We matched content, across two student cohorts, delivered via team-based learning methodology (T cell development and antibody-mediated function) and traditional lecture (B cell development and T cell effector functions). Student learning was assessed using content questions across a range of Bloom’s taxonomy levels, which demonstrated that the TBL activities increased learning. Additionally, students indicated that the TBL activities assisted with preparation for exams and provided a necessary opportunity to address misconceptions. Taken together, TBL intervention increased student performance on basic content, and students found this learning tool to be valuable.
departmental support only
Immune system perturbations in patients with severe long COVID
Authors: Rodriguez Lucie , Tan Ziyang , Lakshmikanth Tadepally , Wang Jun , Barcenilla Hugo , Gonzalez Laura , Mugabo Constantin , Johnsson Annette , Chen Yang , Mikes Jaromir , Landegren Nils , Runold Michael , Bruchfeld Judith , Brodin Petter 
Published: August 2023

Abstract
SARS-CoV-2 infections have different outcomes ranging from mild to severe acute infection as well as post-infectious multi-system inflammatory syndromes (MIS-C/A). Post-acute sequelae of COVID-19 (PASC) or long COVID is another outcome, which is poorly understood and range from mild to very severe persistent organ dysfunction and damages. To maximize our chances of identifying underlying mechanisms of long COVID we focus on the most severe cases with objective measures of disease such as lung damages, microvascular and autonomic nerve dysfunction. We are testing the following hypotheses that I) long COVID is a super-antigen mediated disease, II) an autoimmune condition driven by pathogenic autoantibodies, and/or III) a disease caused by viral persistence. A cohort of over 100 long COVID patients, 24 recovered long COVID patients as well as 9 COVID convalescent controls have been recruited thus far and blood samples analyzed for plasma proteome (Olink assays), immune cell composition and phenotypes (mass cytometry), T-cell repertoire (scTCR-seq), innate cell activation (circulating nucleosomes), and auto-antibody analyses (Luminex arrays) in order to understand immune perturbations in severe long COVID patients. Unlike children with hyperinflammatory MIS-C, we have not found any evidence of superantigen-mediated T-cell activation in long COVID, but instead substantial clonal expansion of memory T cell clones with effector memory states and shared clonality across patients. Additionally, cytokine perturbations such as elevated levels of IL8 and elevated levels of circulating nucleosomes indicate persistent activation of neutrophils in severe long COVID.
Non-fatal outcomes of COVID-19 disease in children with solid organ transplants associates with down-regulation of senescent pathways
Authors: Subramanian Kumar , Tesfai Ivy , Muralidaran Vinona , Seeley Christina , Corbert Lauren , Yazigi Nada , Khan Khalid , Kaufman Stuart , Vitola Bernadette , Matsumoto Cal , Kroemer Alexander , Fishbein Thomas , Ekong Udeme 
Published: August 2023

Abstract

Objective
To study the immune landscape during acute infection (AI) & COVID-19 recovery in children with solid organ transplants (SOTs).

Method
Blood collected between Nov 2020 – June 2022 from pediatric SOT recipients 1–14-days after a positive PCR for SARS-CoV-2 (AI), & 15–60-days after a positive PCR (convalescence), for Ficoll of PBMC. Cryopreserved PBMC taken from pediatric SOT recipients before 2019 used as non-infected controls (ctrls). PBMCs stimulated with 15-mer peptides from spike protein & co-stimulation with anti-CD49d & anti-CD28. Immunophenotyping by mass cytometry. Data analyzed using the Astrolabe Cytometry Platform, which follows a standardized pipeline for population identification & statistics & visualized using the t-SNE dimensionality reduction method. Disease severity stratified using WHO criteria. Subjects unvaccinated against COVID-19.

Results
38 children included: median age & time from transplant (TX) at infection: 5.5 (2.1–15.1) & 3.3 (0.2–14.7)-years for AI (n=10); 2.4 (0.6–15.6) & 0.5 (0.2–11.2)-years for ctrls (n=20); 6.7 (1.0–12.9) & 4.3 (0.2–9.7)-years for convalescence (n=8). NK & CD4+T cell (EM, Th2) frequency increased during AI {p&lt;0.05}. CD57 expression low in T cells with EM phenotype, NKT, & gdT cells in AI vs. ctrls (p&lt;0.05) & in NK cells in severe disease (p&lt;0.05). CD161 expression high in CD4+ T cell (CM, Th1) in convalescence vs. ctrls {p=0.04}.

Conclusion
contraction in NK cells is not a feature of COVID-19 disease in pediatric SOT & global characteristics of NK, NKT & T cell compartments not compatible with senescence. Upregulation of cytolytic effectors in AI. Above a plausible mechanism for low mortality from COVID-19 disease in this patient population.
Structure of pathogenic anti-PF4 autoantibodies reveals the mechanism of immune response formation in vaccine-induced thrombotic thrombocytopenia (VITT)
Authors: Ivanov Daniil G , Ngyuen Son N , Le Si-Hung , Du Yi , Ivetic Nikola , Nazy Ishac , Kaltashov Igor A 
Published: August 2023

Abstract
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe, but rare, side effect of adenoviral-vectored COVID-19 vaccines. It is attributed to formation of autoantibodies recognizing Platelet Factor 4 (PF4) and capable of triggering platelet activation, which leads to thrombosis. We used mass spectrometry (MS) to determine the structure of a VITT patient-derived anti-PF4 antibodies (VITT IgG) and identified their unique properties underlying pathogenicity.
The VITT IgGs studied in this work were extracted from the plasma of a ChAdOx1 vaccine recipient, who developed VITT. Intact-mass MS analysis of these antibodies revealed one major clone, and its complete structural characterization was carried out using MS-based de novo sequencing. This allowed us to establish its subclass (IgG2), determine the amino acid sequence, and identify an N-glycan in the variable region.
This information was used to build a 3D model of the VITT IgG, which revealed the presence of a large polyanionic patch within the paratope involving four CDR regions. Molecular dynamics simulations highlight the role of electrostatics as the major driver of the VITT IgG binding to PF4. The large equatorial belt of the positive charge circumscribing the PF4 tetramer serves as a distributed epitope, allowing it to cross-link up to three VITT IgG molecules, giving rise to large immune complexes capable of FcyRIIa-mediated platelet activation. The molecular mechanism of VITT pathogenesis emerging from this work explains how VITT IgGs and PF4 form large immune complexes in the absence of heparin, which is essential to for platelet activation in a similar disease, heparin-induced thrombocytopenia, and sheds light on the etiology of this devastating condition.
Supported by grant from NIH R01 GM112666
Multi-omic profiling in coeliac disease reveals somatic driver mutations in rogue T cell clones
Authors: Singh Manu , Louie Raymond , Milthopre Claire , Adikari Thiruni , Hardy Melinda , Faulks Megan , Field Matt , Ahlenstiel Golo , Tye-Din Jason , Luciani Fabio , Goodnow Chris C 
Published: August 2023

Abstract
It has long been proposed that the pathogenesis of human autoimmune diseases may share common origins with lymphoid cancer. However, a major hurdle in understanding the immune pathogenesis of autoimmune diseases is distinguishing self-reactive “rogue” lymphocytes from normal immune cells. In Coeliac disease, both the environmental trigger (gluten) and major autoantigen (transglutaminase 2) are well characterised, but the underlying mechanisms by which rogue lymphocytes initiate and drive disease are unknown.
Here, we apply multi-omic technologies that enable detailed DNA, RNA and protein measurements at the single-cell level to profile tens of thousands of immune cells isolated from small intestine duodenum biopsies from individuals with Coeliac disease. We utilise the T-cell receptor as a natural barcode across multiple single-cell experiments to identify expanded rogue T cell clones and their gene and cell-surface protein expression profiles, along with any somatic DNA driver mutations. Strikingly, we identify in multiple patients expanded T cell clones with mRNA and cell-surface protein expression profiles of cytotoxic effector cells, that harboured missense somatic mutations in T cell lymphoma driver genes STAT3, STAT5B and DDX3X. These mutations have been described as strong gain-of-function mutations in multiple T cell lymphomas, highlighting a novel mechanism by which T cell clones escape immune tolerance and adopt a rogue phenotype in Coeliac disease.
Overall, our results highlight the power of single-cell multi-omics in identifying and characterising rare pathogenic clones in a common human autoimmune disease and provide direct evidence for a shared pathogenesis of autoimmunity and lymphoid malignancy.