bluuebunny/crossref_metadata_2025_split huggingface dataset:
TCR signal strength and proliferation affect Runx2 expression and histone modifications in CD8+ T cells
Authors: Michaels-Foster Mary Jessamine , Berg Leslie 
Published: August 2023

Abstract
Runx family transcription factors are crucial for the establishment of genetic programs during important cell differentiation stages. Although the roles of Runx1 and Runx3 in T cells have been extensively studied, the role of Runx2 has not fully been explored. T cell specific Runx2 deletion leads to a cell intrinsic defect in the longevity of the CD8+ memory population and decreased expression of memory markers like TCF1 and Eomes. As histone modifiers are known Runx2 binding partners, we propose that Runx2 may bind CD8+ memory associated gene promotors, allowing for the maintenance of chromatin as memory cells undergo homeostatic proliferation. To further explore the function of Runx2 in T cells, we utilize a Runx2 fl/fl CD4-Cre model of T cell specific Runx2 deletion. We use an in vitro CD8+ T cell stimulation assay in which we co-culture DC2.4 (a mouse dendritic cell line) cells loaded with ovalbumin peptide variants (N4, T4, and V4) with OT-1 CD8+ T cells for 72 hours to observe the effects of Runx2 expression changes. Additionally, we utilize chromatin analyses in this system to examine histone modifications associated with Runx2 activity. In accordance with previous data, Runx2 expression is inversely correlated with TCR signal strength, as cells stimulated with low dose/affinity peptides upregulate higher levels of Runx2. Additionally, cell proliferation dye labeling reveals that Runx2 expression increases as cells divide during stimulation and continues to increase as activated CD8+ T cells are rested post-stimulation. Preliminary data also suggest that histone modifications associated with poised chromatin vary with Runx2 expression, indicating that Runx2 may play a role in chromatin modifications in CD8+ memory T cells.
Single-cell multi-omic analysis reveals serum amyloid A3 restrains alternative activations in macrophages derived from different origins
Authors: Lin Jian-Da , Chou Tzu-Yin , Ye Yu-Zhen , Lin I-Jung , Lien Chia-Jung , Wang Zhih-Yuan , Yang Chen-Hsuan , Chao Pei-An , Chen Yen-Ting , Loke P’ng 
Published: August 2023

Abstract
Alternatively activated macrophages (AAMs) play pivotal roles in regulating metabolism, anti-helminth infections, and tissue homeostasis. AAMs derived from different cellular origins are functionally and phenotypically distinct, but the regulatory networks mediating these differences remain unclear. Here, we use a combination of CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing; a 128 or 198 barcode-antibody panels) and genetic fate mapping, to profile F4/80int CD206+ macrophage (Mφ) and F4/80hi CD206-tissue-resident Mφ derived from CX3CR1+ precursors, in mice during homeostasis and Interleukin 4 (IL-4) or Heligmosomoides polygyrus initiated alternative activation. The analyses reveal greater complexity in embryonic-derived F4/80hi CD206-tissue-resident Mφ than F4/80int CD206+ Mφ or Mφ with tissue-resident phenotype from monocyte origins during homeostasis. We show that serum amyloid A3 (Saa3) is exclusively expressed in tissue-resident Mφ than other immune cell types and negatively associated with typical M2 markers such as Retnla, Mrc1, and Mgl2 expression in Interleukin 4 (IL-4) or Heligmosomoides polygyrus (H. Poly) initiated AAMs. The significant expansion of Retnla+ Mφ is observed in H. Poly- or IL-4 treated Saa3−/− mice and presents less worm burden and more M2 metabolic phenotypes than WT mice. Notably, we also identified a Folr2+ tissue-resident Mφ population that constitutively expresses Retnla and is associated with leukocyte and T-cell activation. Hence, single-cell multi-omic analysis allowed us to identify novel regulatory networks and distinct Mφ properties from different origins during alternative activation.
Deciphering the role of platelet alpha granules in alveolar-capillary breakdown in severe malaria
Authors: Reed Jenna , Deckers Margot , Van den Steen Philippe , Petrey Aaron , Evavold Brian D , Lamb Tracey 
Published: August 2023

Abstract
Malaria is considered the deadliest disease in human history, killing hundreds of thousands annually. A hallmark of severe malaria is the breakdown of blood-organ barriers, such as in the lungs and brain, but the precise mechanism of how this occurs remains unknown. We determined that, unlike wildtype mice (C57BL6), mice with platelet alpha-granule deficiencies (Nbeal2−/−) did not die of malaria-associated acute respiratory distress syndrome (MA-ARDS) when infected with Plasmodium berghei NK65-Edinburgh (PbE). While wildtype mice exhibited significant alveolar-capillary breakdown by 6 days post-infection, as measured by alveolar protein concentrations and Evans blue permeation, Nbeal2−/− mice did not develop severe pathology. Flow cytometric analysis revealed that PbE-infected Nbeal2−/− mice had reduced CD8 T cell lung recruitment and activation compared to wildtypes. From this data, we concluded that a component of platelet alpha granules is crucial for activating CD8 T cells that subsequently damage the alveolar barrier. We hypothesized that the enzyme hyaluronidase 2 (HYAL2) is that crucial component and plays a 2-fold role: 1) that it directly contributes to barrier breakdown by cleaving hyaluronan (HA) from the endothelial glycocalyx (eGC), and 2) that the HA fragments released after eGC cleavage promote inflammation via CD44-binding. We found that HA is elevated in the plasma of infected mice. Preliminary data suggest that CD44 splice variants are differentially expressed in the lungs of naïve vs. PbE-infected mice, which could alter the binding kinetics with HA. Together, these data support the hypothesis that platelet HYAL2 is a critical mediator of MA-ARDS.
Novel murine model reveals an early role for pertussis toxin in disrupting neonatal immunity to Bordetella pertussis
Authors: Sedney Colleen , Caulfield Amanda , Dewan Kalyan , Callender Maiya , Manley Nancy , Harvill Eric T 
Published: August 2023

Abstract
The increased susceptibility of neonates to some pathogens has previously been attributed to an underdeveloped immune system. More recent data suggest neonates have effective protection against most pathogens but are particularly susceptible to those that target immune functions specific to neonates. Bordetella pertussis (Bp), the causative agent of “whooping cough”, causes more serious disease in infants attributed to its production of pertussis toxin (PTx). Problematically, the rapid development of adult immunity in mice has confounded our ability to study the interaction of this agent with neonatal immune components, such as virtual memory T cells which are prominent prior to the maturation of the thymus. Here, we examine the rapid change in susceptibility of young mice and define a period from five- to eight-days-old during which mice are much more susceptible to Bp than mice even a few days older. These more narrowly defined “neonatal” mice display significantly increased susceptibility to wild type Bp but very rapidly and effectively respond to and control Bp lacking PTx, more rapidly even than adult mice. The clearance of the PTx mutant correlates with the early accumulation of neutrophils and T cells and suggests a role for PTx in disrupting their accumulation. These results define an early age of extreme susceptibility to Bp, and demonstrate that the neonatal response can be more efficient than the adult response in eliminating bacteria from the lungs, but these neonatal functions are substantially blocked by PTx. This refined definition of “neonatal” mice may be useful in the study of other pathogens that predate neonates, and PTx may prove a particularly valuable tool for probing the poorly understood neonatal immune system.
This work was supported by grants AI156293 and AI159347 of the National Institutes of Health to Eric Harvill and AI139449 of the National Institutes of Health to Nancy Manley.
Targeted activation of IL-22 producing innate lymphoid cells enhances host resistance against Clostridioides difficile infection
Authors: Abt Michael C , Denny Joshua E , Maslanka Jeffrey , Mdluli Nontokozo V , Mears Kevin S 
Published: August 2023

Abstract
Clostridioides difficile, the most common hospital acquired pathogen in the United States, infects the gastrointestinal tract following perturbation of the intestinal microbiota causing debilitating, potentially fatal colitis. Primary C. difficile infection has a high recurrence rate following antibiotic treatment, emphasizing the need to develop alternative treatment strategies to reduce the burden on the healthcare system. Innate lymphoid cells (ILCs) are necessary for acute host defense following C. difficile infection and represent a promising therapeutic target to harness the patient’s innate immune defenses to limit disease. Here, using a murine model of C. difficile infection, we report that oral administration of Resiquimod (R848), a TLR-7 agonist previously reported to activate type-3 ILCs (ILC3s), protects mice from lethal C. difficile challenge. R848 treatment did not alter intestinal microbial communities or impact C. difficile burden. R848 treatment conveyed protection via activation of the host immune system in a TLR-7 dependent manner. R848-mediated protection was independent of T and B cells as well as type-I IFN signaling. Mice lacking ILCs, however, did not respond to R848 treatment and remained susceptible to infection, identifying these cells as the key targets of R848 activity. Last, R848 treatment robustly induced ILC3s in the large intestine to produce IL-22 and genetic ablation of IL-22 abrogated R848-mediated protection from C. difficile infection. Combined, these data identify an immunostimulatory molecule that activates the IL-22 producing ILC3s to support host defense against C. difficile infection without causing further dysbiosis of the intestinal microbiota.
NIH R01AI158830, R21AI164385
Understanding Asymptomatic Malaria: The Utility of Wild-Derived Mouse Diversity
Authors: Cornwall Douglas , Srey Mellina T , Heins Kate , Maloba Franklin , Evavold Birk , Olatunde Adesola , Fogang Balotin , Jensen Anne , Ayong Lawrence , Evavold Brian D , Lamb Tracey J 
Published: August 2023

Abstract
With over 200 million clinical cases per year leading to over 600,000 deaths, malaria is still a significant global health problem. Up to 80% of individuals in some malaria-endemic areas can have asymptomatic carriage of Plasmodium infections (no overt symptoms) and are thought to be a significant reservoir for malaria transmission. Many asymptomatic Plasmodium-infected individuals show mild anemia and vascular activation and have an increased risk for co-morbidities such as typhoidal Salmonella. Currently, the immunological mechanisms governing asymptomatic Plasmodium carriage are poorly understood, partly due to a lack of immunologically intact asymptomatic animal models. Here, utilizing a unique genetically diverse SPF wild-derived mouse colony we address the immunological underpinnings of asymptomatic malaria. This model shows that asymptomatic Plasmodium carriage is genetically controlled. Furthermore, wild-derived mice have varying levels of anemia associated with non-lethal Plasmodium yoelii XNL infection; some mice exhibit an asymptomatic phenotype with mild anemia independently of parasitemia. Anemia levels correlate with the production of TNF-a and IL-10, which is consistent with patterns seen in asymptomatic children in Cameroon. CD4 T cells producing IL-10 and IFN-g were also correlated with the severity of anemia, with few T regulatory cells that expanded during infection, consistent with the pattern of circulating CD4 T cells responses seen in pediatric asymptomatic malaria. Previously unknown genes are also found in GWAS analysis in these mice. Our results highlight that this novel model can be used to determine the immune mechanisms that underpin the asymptomatic carriage of Plasmodium parasites.
Supported by a grant from NIH (R01AI167422)
Distinct 3D remodeling of Il4-Il13-Il5 loci mediates differential type 2 responses in innate versus adaptive lymphocytes
Authors: Nagashima Hiroyuki , Shayne Justin , Kanno Yuka , O’Shea John J 
Published: August 2023

Abstract
The type 2 cytokines, interleukin (IL)-4, IL-5 and IL-13 reside within a tandem multi-gene cluster in mammals. These cytokines represent the hallmark of type 2 immune responses controlling parasites, promoting tissue repair as well as causing allergic diseases. Both innate and adaptive lymphocytes secrete type 2 cytokines with discordant production spectra. However, how those two related but distinct type 2 lymphocytes configure the extended type 2 cytokine loci and elicit selective output of this cassette genes is not well understood. Here, we took a holistic structural and functional view of the type 2 cytokine locus before and after activation, comparing innate (ILC2) and adaptive (Th2) lymphocytes to understand mechanisms underlying their distinctive programs. Rapid induction of IL-5 dominates in ILC2, whereas IL-4 does so in Th2 cells. Using high-resolution chromatin conformation capture we found that global cellular chromatin architecture remained constant, whereas the type 2 cytokine locus rapidly remodeled. In ILC2, Il13 and Il5 loci were aligned in proximity whereas Il4 locus was insulated. In Th2 cells, Il4 and Il13 positioned in proximity while the Il5 locus remained distal. Select REs were separately deleted in mice to confirm cell-type specific and activation-dependent roles in type 2 responses in vivo. Thus, contrary to the premise that chromatin architecture plays a minimal role in steady-state gene induction, signal-dependent remodeling of 3D configuration underlies the discordant cytokine outputs in ILC2s versus Th2 cells.
Bottoms up!: Inferring relevant immune phenotypes from bulk cytokine kinetics via semi-supervised machine learning
Authors: Krishnan Anagha Yogam , Wahlsten Madison , Lau Vivian , Gerard Audrey , Altan-Bonnet Grégoire 
Published: August 2023

Abstract
Immunologists frequently collect data on continuous multi-dimensional single-cell characteristics then classify cells based on these characteristics. Often, relevant cellular phenotypes (clusters) are determined by an iterative, intuition-driven process that requires significant biological validation. This begs the question: can we efficiently predict immunologically-relevant phenotypes by directly linking them to the biological question we are asking?
To that end, we developed a semi-supervised machine learning algorithm that directly links multi-parametric single cell data to bulk cytokine kinetics. Using T cells primed by antigen-presenting tumor cells as a biological model system, we measured bulk cytokine levels and single-cell surface marker expression every 6 hours for 72 hours. To aid with experiments, we developed a novel protocol for automated sample collection and processing. From these samples, we constructed detailed fitted time kinetics of three cytokines: interferon-gamma (IFNg), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNFa). By training a semi-supervised algorithm on these kinetics, we determined an optimal set of phenotypes that explained cytokine production by T cells. When we applied our model to novel experimental systems (varying the effector:target ratio of cells, changing the peptide quality, etc.), we predicted the resultant cytokine levels and found phenotypes relevant to early shutdown of cytokine production in large, immunosuppressive tumors.
Our algorithm serves as a broadly applicable method to directly link single-cell phenotypes to bulk biological data. We aim to use this method to quickly identify phenotypes of interest for a variety of immunological questions.
Supported by the NIH Oxford-Cambridge Doctoral Fellowship and the Cambridge Trust Fellowship.
Tissue-specific determinants of the adaptive NK cell response
Authors: Mujal Adriana M , Owyong Mark , Santosa Endi K , Wingert Claire , Grassmann Simon , Lau Colleen M , Sun Joseph Chai-Yuen 
Published: August 2023

Abstract
Natural killer (NK) cells are circulating innate lymphocytes that are poised to respond rapidly upon encounter with cancer or virally-infected cells as well as proinflammatory cytokines. In addition to their early role in immune responses, it is now appreciated that NK cells can also take on features of adaptive lymphocytes such as antigen-specific clonal expansion and long-lived memory responses, with these processes best characterized following cytomegalovirus (CMV) infection in mouse and human. NK cell receptor recognition of virally-encoded proteins drives CMV-specific NK cells, but the signals that collectively give rise to these adaptive NK cells are still not fully understood. Mouse cytomegalovirus (MCMV) infection is systemic and while NK cells exhibit cytokine-induced activation across multiple tissue sites of infection, we were surprised to find that MCMV-specific NK cell activation and expansion occurs predominantly in the spleen. Transcriptional analysis indicated that canonical and non-canonical NF-kb components may be engaged to a greater extent in NK cells primed in the spleen, and we further identified TNFa signaling as a critical regulator of both innate and adaptive NK cell responses through engagement of distinct downstream signaling arms. Notably, TNFR2-deficient NK cells exhibited hyper-activation and ultimately failed to undergo the strong proliferative burst we observed in the spleen. These findings highlight the central role of the spleen in facilitating the innate-to-adaptive transition NK cells undergo and provide insight into how we can better generate adaptive NK cell immunity across diverse settings.
Adriana M. Mujal is supported by the Cancer Research Institute as a Cancer Research Institute/Amgen Fellow.
Synergy of heterodimeric IL-15 immunotherapy and a Fatty Acid Metabolism Modulator enhances intratumoral lymphocyte metabolism and eradicates EO771 murine breast tumors
Authors: Karaliota Sevasti , Stellas Dimitris , Stravokefalou Vasiliki , Myers Breana , Felber Barbara K , Pavlakis George N 
Published: August 2023

Abstract
Tumor-infiltrating cytotoxic CD8+ T cells frequently acquire an altered state of differentiation referred to as “exhaustion”, which prevents control of tumor growth. We study hetIL-15, a cytokine that stimulates the generation, proliferation and cytotoxic function of CD8+ T and NK cells. The objectives of this study was to assess the effects of hetIL-15 immunotherapy in TNBC tumors, to evaluate the metabolic profile of the tumor-infiltrating T cells and to study any potential synergy with a Fatty Acid Metabolism Modulator (FAMM). Using the EO771 orthotopic breast cancer model we monitored the efficacy of locoregional administration of hetIL-15 in combination with a FAMM. We monitored the effect of treatment on tumor growth, survival of mice and the status of tumor-infiltrating immune cells using Seahorse and flow cytometry. hetIL-15 locoregional administration resulted in complete tumor regression in 40% of the treated animals and increased survival. The infiltrating cytotoxic CD8+ T and NK cells were increased in hetIL-15 treated tumors and showed enhanced activation and proliferation. Their metabolic flux analysis confirmed a robust rise in Oxygen Consumption Rate (OCR) with substantial increase of spare respiratory capacity. Combined treatment of hetIL-15 immunotherapy and FAMM resulted in increased mitochondrial function, FA uptake and OCR. In addition, combination therapy resulted in statistically significant tumor growth delay and complete eradication of the tumors in 85% of mice. Our results indicate that metabolic reprogramming of tumor-specific CD8+ T cells might represent a strategy to promote survival in the metabolically hostile Tumor Microenvironment (TME), enhancing the clinical efficacy of immunotherapy.
Circulating cell-free DNA directly inhibits T cell function and contributes to peripheral immunosuppression in experimental GBM
Authors: Ayasoufi Katayoun , Wolf Delaney M , Reesman Rachael A , Rangel Frances A , Gulbicki Lauren R , Tritz Zachariah , Jin Fang , Hansen Michael , Fain Cori E , Pfaller Christian K , Johnson Aaron J 
Published: August 2023

Abstract
Glioblastoma (GBM) is an incurable brain cancer that is associated with severe peripheral immunosuppression. This immunosuppression is a critical barrier to patient survival and the success of immunotherapies. C57BL/6 mice harboring GL261 gliomas present with the hallmark features of peripheral immunosuppression observed in GBM patients, including T cell lymphopenia. We determined that peripheral immunosuppression involves release of non-steroidal soluble factors with large molecular weight that are found in sera of glioma-bearing mice. Sera of glioma-bearing mice inhibits T cell proliferation ex vivo. To determine the identity of the immunosuppressive molecules in serum, we used two parallel approaches: 1.) evaluation of abundant macromolecules and 2.) proteomics analysis using tandem mass tag mass-spectrometry (TMT-MS). We determined that serum of glioma-bearing mice contains significantly higher levels of cell free DNA (cfDNA) compared to controls. TMT-MS identified histones as a highly abundant protein in serum, further implicating DNA-histone complexes as top candidates. DNAse treatment of sera isolated from GL261 glioma-bearing mice reduced the suppressive effect on T cell proliferation. We next performed T cell proliferation assays in the presence of pure cfDNA isolated from sera of glioma-bearing mice while genomic DNA was used as control. cfDNA from serum potently inhibited T cell proliferation while control genomic DNA did not. These results were AIM2 independent implicating a novel pathway of DNA sensing in T cells. We contend that cfDNA induces immunosuppressive effects in experimental GBM. Devising strategies to reverse immunosuppression through targeting cfDNA could improve outcomes in GBM patients.
Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ) and Brains Together for a Cure Foundation (KA).
Detection of PD-1 for flow cytometry immunophenotyping using commercial antibodies in clinical studies with check point inhibitors
Authors: Bisconte Angelina R , Mohanram Venkat , Yang Ming , Phippard Deborah 
Published: August 2023

Abstract
Precision for Medicine (Precision) has optimized the use of commercial anti-PD-1 and anti-IgG4 reagents to detect both free PD-1 and/or Pembro-bound PD-1 in pharmacodynamic studies when Pembrolizumab, a concomitant check point inhibitor, is used in combination with a new therapy. Precision has developed and qualified flow immunophenotyping assays to quantify PD-1 on CD4+ and CD8+ T cells using a commercial PD-1 clone (PD1.3.1.3) and an anti-IgG4. This enabled detection of both free and drug bound PD-1 receptor. We compared detection of PD-1 in PHA stimulated healthy donors treated with saturating Pembrolizumab or no drug treatment. There was not a significant difference observed in PD-1 quantification in the presence or absence of Pembrolizumab, which demonstrated that our approach was able to accurately measure total PD-1 whether free or bound. As Pembrolizumab was titrated from the saturating concentration there was a dose-dependent decrease in detection of PD-1. Here we will demonstrate the detection of PD-1 using the above methodology compared to other commercial PD-1 antibody clones. In clinical trials which incorporate checkpoint inhibitors like either PD-1 or PD-L1, the selection of commercial reagents are key considerations to detect these receptors when designing a sensitive, selective, and accurate biomarker assay.