bluuebunny/crossref_metadata_2025_split huggingface dataset:
CD4 and CD8 double positive T cell progenitors in mediastinal adipose tissue and the aortic arch differentiate to CD4 T cells
Authors: Winkels Holger , Ghosheh Yanal , Kobiyama Kouji , Kiosses William B , Marcovecchio Paola , Orecchioni Marco , Wolf Dennis , Thiault Nicolas , Seumois Gregory , Vijayanand Pandurangan , Ley Klaus 
Published: January 2023

Abstract
Using 10× Genomics-based single-cell RNA sequencing on digested atherosclerotic mouse aortas, we discovered an unexpected immune cell population whose transcriptome was similar to thymic CD4+CD8+ cells (DPs). Flow cytometry showed DP presence in the aortic arch (AA) and mediastinal adipose tissue (MAT), but not in lymph nodes, spleen, blood or other tissues. DPs in MAT or AA did not change with atherosclerosis in Apoe−/− mice. Fluorescence microscopy of cleared whole mounts placed DPs in the AA adventitia and near microvessels in MAT. MAT and AA DPs were absent in Rag2−/− mice and in day3 and 21 thymectomized mice. Deep transcriptional profiling from sorted thymic, MAT, and AA DPs showed hundreds of differentially expressed genes. While thymic DPs were enriched for TCR signaling pathways, extra-thymic DPs were enriched for stress responses and cell cycle pathways. Pulse-chase experiments demonstrated a five-day turnover of MAT and AA DPs. Functionally, sorted MAT DPs developed successfully into conventional T cells (with a bias to CD4) in hanging drop cultures. Pharmacologic inhibition of cell migration by S1P1 blockage with FTY720 did not affect the presence of these cells, suggesting that migration of extra-thymic DPs does not require passage into blood. Transplantation of congenically marked thymi under the kidney capsule of thymectomized mice reconstituted T cells in the inguinal lymph node, but failed to reconstitute extra-thymic DPs. The discovery of two new, transcriptionally distinct subsets of extrathymic DPs reveals a novel, unexpected local escape mechanism from the thymus. Their association with blood vessels suggests possible angiotrophic functions.
THEMIS functions as a cytoplasmic adaptor and nuclear transcription factor in developing thymocytes and mature T cells
Authors: Thiault Nicolas , Kakugawa Kiyokazu , Takazawa Ikuo , Verstichel Greet , Taniuchi Ichiro , Cheroutre Hilde 
Published: January 2023

Abstract
THEMIS functions in the cytoplasm as an adaptor molecule which dampens TCR signaling. During thymic development, THEMIS sets the affinity threshold for activation, enabling the selection of conventional T cells in response to low affinity antigens. Although these events occur in the cytoplasm, THEMIS possess a nuclear localization signal (NLS) and a significant amount of THEMIS resides in the nucleus.
To investigate the importance of THEMIS in the nucleus, we deleted the Themis NLS to force THEMIS in the cytoplasm. Surprisingly, T cell development was impaired similar to the Themis null mice, suggesting that THEMIS may have important roles in the nucleus as well. Alternatively, we established Themis mutant mice, in which THEMIS localized exclusively in the nucleus or where THEMIS was constantly present in both compartments. Normal conventional T cells were absent in both mutant mice. Since lack of THEMIS or the constant presence of THEMIS in the cytoplasm or nucleus or both, all resulted in aberrant T cell development it indicated that the translocation of THEMIS to or/and from the nucleus is essential for its function.
To examine the role of THEMIS in T cells in the periphery, we generated mutant mice where THEMIS is expressed normal during thymic development but altered post-thymically. Adoptive transfer of naïve CD4 T cells with altered THEMIS expression into RAG deficient host induced enhanced sever colitis and furthermore, we observed that forced localization of THEMIS in the nucleus redirects the polarization of CD4 T cells toward the pathogenic TH17 helper lineage.
These findings identify a dual function for THEMIS as a cytoplasmic adaptor during TCR signaling and a nuclear factor directing gene expression in thymocytes and mature T cells.
Cellular requirements for B7 expression in thymic iNKT cell development
Authors: Watanabe Masashi , Celli Sofia , Alkhaleel Farrah , Hodes Richard J 
Published: January 2023

Abstract
We had previously shown that the B7-CD28 co-stimulatory pathway is important for thymic iNKT cell development. iNKT cell development is dependent on glycolipid/CD1d complex presented by CD4+ CD8+ double positive thymocytes, with no requirement for CD1d-expression on other cell types, including thymic epithelial cells (TEC), dendritic cells (DC), or B cells. However, the identity of the specific cell type(s) providing B7 co-stimulation for developing thymic iNKT is unknown. Here we have analyzed the cellular requirement for B7 expression utilizing B7 conditional KO approaches. We found that B7 expression on TEC, DC and B cell have essential but redundant roles in supporting thymic iNKT cell development. These results suggest that during thymic iNKT cell development, signal 1 (glycolipid/CD1d-TCR) and signal 2 (B7-CD28) are provided by spatially and temporally distinct cell types and may provide new insight into how co-stimulation can function in immune system development.
Loss of Dll4 expression in thymic epithelial cell monolayer culture is mediated by reduction in both burst size and burst frequency of its gene transcription
Authors: Han Jianxun , Kianizad Korosh , Zúñiga-Pflücker Juan Carlos 
Published: January 2023

Abstract
Commitment of thymic seeding progenitor (TSP) cells to the T-lineage depends on Notch signaling, which requires engagement of the Notch1 receptor on TSPs by the Notch ligand Dll4 on thymic epithelial cells (TECs). We previously showed that when TECs are cultured as monolayers (2-D), Dll4 expression is rapidly lost, as is their ability to support T-cell development in vitro. In the present study, we aim to examine the regulation of Dll4 expression in TECs at the levels of gene transcription and mRNA degradation in TECs from E15 embryos using a combination of multiple techniques. The half-life of Dll4 mRNA in TECs was found, by utilizing quantitative PCR (qPCR) combined with Uridine RNA analog pulse-labeling, only approximately 30 min, which makes its level very sensitive to changes in the rates of gene transcription. Nonetheless, reduction in gene transcription, rather than in mRNA stability, was determined to be the primary molecular mechanism underlying the loss of Dll4 expression in 2-D TECs. FoxN1, the transcription factor that drives thymic epithelial cell development, was found to bind to multiple sites at the Dll4 gene promoter and an enhancer region within its third intron by luciferase reporter assay. Consistent with the luciferase reporter assay result, enforced expression of FoxN1 in 2-D TECs restored Dll4 expression significantly. Further detailed analysis with PrimeFlow RNA assay revealed that FoxN1 regulates only the burst size, but not the burst frequency, of Dll4 gene transcription. We are currently working on identifying the transcription regulator(s) that could bind to the enhancer region to regulate the burst frequency of Dll4 gene transcription.
Bone marrow-derived cells contribute to the maintenance of thymic stroma including thymic epithelial cells
Authors: Chakrabarti Shami , Hoque Mohammed , Jamil Nawshin Zara , Meer Neelab , Singh Varan J , Pezzano Mark T 
Published: January 2023

Abstract
In paradox to critical functions for T-cell selection and self-tolerance, the thymus undergoes profound age-associated atrophy and loss of T-cell function, which are further enhanced by cancer therapies. Identification of thymic epithelial progenitor populations capable of forming functional thymic tissue will be critical in understanding thymic epithelial cell (TEC) ontogeny and designing strategies to reverse involution.
We identified a new population of progenitor cells, present in both thymus and bone marrow (BM), that co-express the hematopoietic marker CD45 and the definitive thymic epithelial marker EpCAM and maintains the capacity to form functional thymic tissue. Confocal analysis and qRT-PCR of sorted cells from both BM and thymus confirmed co-expression of CD45 and EpCAM. Grafting of C57BL/6 fetal thymi under the kidney capsule of H2BGFP transgenic mice revealed that peripheral CD45+EpCAM+GFP+ cells migrate into the developing thymus and contribute to both TECs and FSP1-expressing stromal cell. Sorted BM-derived CD45+EpCAM+ cells contribute to reaggregate thymic organ cultures (RTOCs) and differentiate into keratin and FoxN1 expressing TECs, demonstrating that BM cells can contribute to the maintenance of TEC microenvironments previously thought to be derived solely from endoderm.
BM-derived CD45+EpCAM+ cells represent a new source of progenitor cells that contribute to thymic homeostasis. Future studies will characterize the contribution of BM-derived CD45+EpCAM+ TEC progenitors to distinct functional TEC microenvironments in both the steady-state thymus and under conditions of demand. Cell therapies utilizing this population may prove useful for counteracting thymic involution in cancer patients.
A novel dual-reporter lineage tracing model reveals phenotypic and functional heterogeneity of tissue-resident macrophages
Authors: Eddins Devon James , Kosters Astrid , Ghosn Eliver E.B. 
Published: January 2023

Abstract
Tissue-resident macrophages (TRMΦ) are important immune sentinels responsible for maintaining tissue and immune homeostasis, where most develop during embryogenesis independently of long-term hematopoietic stem cells (LT-HSCs) and are maintained throughout life with minimal contribution from circulating monocytes. Recent studies have shown that, in certain conditions, infiltrating monocyte-derived (MD)MΦ can supplement TRMΦ, but remain distinguishable. However, the phenotypic and functional heterogeneity of TRMΦ versus their supplemented MDMΦ counterparts in each organ system has not been established. To address this gap, we have generated a novel dual-reporter lineage tracing model to delineate TRMΦ vs MDMΦ in vivo. We used a non-genotoxic method of HSC depletion in the Runx1Cre/eGFP mouse, which allows us to transplant LT-HSCs expressing mRFP1. These dual-reporter mice exhibit normal supplementation of MDMΦ (red) to the existing TRMΦ (green) pool. We have previously identified two subsets of peritoneal MΦ that exhibit dual ontogeny. However, an in-depth examination of the phenotypic and functional differences between these two MΦ lineages is lacking. Using our new model, we performed single-cell mRNA sequencing and high-dimensional flow cytometric analyses and reveal further heterogeneity in peritoneal MΦ across lineages, supporting the notion that infiltrating MDMΦ exhibit phenotypic divergence from TRMΦ with potential functional impact. Here, we report the generation and utility of an innovative model incorporating lineage-specific fluorescent reporters in radiation-naïve mice, an important tool for understanding phenotypic and functional diversity of MΦ with different developmental origins in vivo.
Identification of a CD117+CD71+ early neutrophil progenitor population in human bone marrow
Authors: Dinh Huy , Eggert Tobias , Meyer Melissa , Zhu Yanfang , Olingy Claire , Llewellyn Ryan , Wu Runpei , Hedrick Catherine C. 
Published: January 2023

Abstract
Neutrophils play critical roles in health and disease. Due to their very short half-life in blood and tissue, neutrophils are constantly replenished by bone-marrow progenitors. Thus, a comprehensive understanding of bone marrow neutrophil development is of paramount importance to identify how neutrophil production is altered in disease. Recently, two novel human neutrophil progenitor populations were identified; ‘human neutrophil progenitor’ or ‘hNeP’ (Lin-CD66b+CD117+) and ‘neutrophil precursor’ or ‘preNeu’ (Lin-CD66b+CD15+CD49d+). How these subsets fit into the neutrophil lineage is unclear. By using mass and flow cytometry, we show that hNeP are a heterogenous population containing a homogeneous progenitor subset termed ‘early neutrophil progenitor’ or ‘eNeP’ (Lin-CD66b+CD117+CD71+). Surface marker and RNA expression, together with the ability to form colonies in vitro suggest that eNePs, which constitute only ~0.14% of bone marrow neutrophils, are upstream of preNeu (~5% of bone marrow neutrophils). Furthermore, we have identified novel neutrophil surface markers associated with distinct developmental stages, including CD71. Intriguingly, CD71+ characterizes proliferating neutrophils, which are expanded in the blood of melanoma and lung cancer patients and detectable in human lung tumors. Collectively, our findings i) identify CD117+CD71+ eNeP as an early neutrophil progenitor population, ii) introduce a unified model of human neutrophil bone marrow development, iii) identify novel surface markers for distinct neutrophil developmental stages and iv) provide evidence for neutrophil progenitor expansion in cancer.
Mast cells as Immune modulators of behavior and brain cellular composition
Authors: Duque-Wilckens Natalia , Zweng Sonja , Teis Robert , Robison AJ , Moeser Adam J 
Published: January 2023

Abstract
Mast cells (MC) are involved in peripheral disease such as allergy/anaphylaxis, but they are also present in brain where they can modulate behavior. MC deficient KitW–sh/W–sh (sash) mice have been reported to show increased anxiety-like behaviors compared to wild type (WT) mice, but these studies did not consider potential interactions with sex and/or stress, which is relevant considering that MCs are sexually dimorphic and stress-sensitive. Here, wild type (WT) and sash male and female mice were exposed to control handling or mild environmental stress and then phenotyped for behaviors relevant to human mood disorders. Stress did not alter anxiety or motivated behaviors in WT. Compared to WT, sash showed increased anxiety, which was independent of sex or stress. A measure of depressive-like behavior in rodents is preference for sucrose water over regular water. In females, sash showed reduced sucrose preference compared to WT, which was exacerbated by stress. In males, control sash and WT showed similar sucrose preference, but stress induced sucrose anhedonia only in sash. To explore the underlying mechanisms, we studied the Nucleus Accumbens, a brain area involved in anxiety and motivation. We found that sash have a reduced number of astrocytes and a two-fold higher number of cells expressing FosB, a transcription factor induced by stress and involved in synaptic plasticity and behavior. Together, these data implicate MCs as key immune modulators of brain cellular composition and activity. Our ongoing investigations aim at deeply understanding the role that MCs play on mood and cognition, which could provide significant insights into the mechanisms underlying human psychiatric disease and inform new therapeutic targets.
Contribution of CD137-CD137L pathway in sciatic nerve crush-induced behavioral hypersensitivity and functional reduction
Authors: Wakley Alexa A , Cao Ling 
Published: January 2023

Abstract
The T cell costimulatory molecule CD137L is involved in the development of neuropathic pain following peripheral nerve injury. Previously, our work has shown that absence or neutralization of CD137L reduces hypersensitivity, sensory and functional impairment following a sciatic nerve crush (SNC). CD137L activation utilizes both a CD137-dependent and a CD137-independent pathway. To further determine the contribution of these pathways, we examined pain-like and functional recovery-related behavioral responses following intrathecal injection (IT) of a CD137 neutralizing antibody (αCD137; daily days 3–10) after SNC in male and female B6 mice. Mice were randomly assigned to 1 of 4 groups: PBS, 0.5μg isotype control, 0.1 or 0.5μg of αCD137, and then were evaluated for sensitivity (von Frey, Hargreaves, & cold plantar test), sensory (pin prick assay) and motor (hind limb grip strength, toe spread reflex and toe spacing score) functions before surgery and on post-surgery days 1–77. On post-SNC day 1, all groups showed changes in sensitivity and decreased sensory and motor functions. αCD137 treatment did not alter mechanical, heat or hypersensitivity compared to PBS- or isotype-treated controls. αCD137-treated groups showed faster sensory (pin prick score) and motor functional recovery (toe spread reflex, toe spacing), as well as slightly greater improvement in grip strength test compared to controls. Together, our results indicate partial involvement of the CD137-dependent pathway in SNC-induced sensory and motor function changes following peripheral nerve injury. (Supported by NIH/NINDS R01NS098426 (Cao))
Immune response reflecting the involvement of Complement components in Gulf War illness
Authors: Brahmajothi Mulugu V 
Published: January 2023

Abstract
Gulf War Illness (GWI) encompasses multisymptom disorders characterized by chronic fatigue exhibiting neuromuscular involvement, affecting a considerable number of veterans who served the Persian Gulf War between 1990–91. Every war brings home soldiers with some acute/chronic disorders, but this multisymptom illness categorized as GWI is being investigated for 28 years because the treatments have been only palliative in spite of the considerable effort to treat the veterans to wellness. There are still unmet needs in understanding the pathobiology of GWI. Several studies conducted so far attribute the cause to chemical and biological exposures. From in-vivo studies, it can be deduced that the combined repetitive exposures to various neurotoxic chemicals insistently altered cellular receptors. Further, prophylactic treatments such as pyridostigmine bromide, anti-nerve gas pills, glucocorticoids, corticosterone, and vaccines not only primed the immune system but also paradoxically triggered the CNS-glymphatic system to mount a neuroinflammatory response in some individuals. This study focused on relative estimates of complement components and the convertases in the plasma, considering the immune system is the primary defense consisting of high polymorphism combined with a wide range of soluble factors. Complement components from C1 to C9 show high variability in 25 veterans with GWI compared to 20 healthy controls and other referenced neuromuscular disorders. Although C1 is the first responder of the classical pathway, consistently and significantly C1q, C5b, C7, factor D, and properdin were upregulated with the highest variability in veterans with GWI (p&lt;0.001), indicating distinct variance in the immune complex.
Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and -independent cellular responses in Alzheimer’s disease
Authors: Zhou Yingyue , Song Wilbur M. , Andhey Prabhakar , Swain Amanda , Levy Tyler , Miller Kelly R , Poliani Pietro L. , Cominelli Manuela , Grover Shikha , Gilfillan Susan , Cella Marina , Ulland Tyler K , Zaitsev Konstantin , Miyashita Akinori , Ikeuchi Takeshi , Sainouchi Makoto , Kakita Akiyoshi , Bennett David A , Schneider Julie A , Nichols Michael R , Beausoleil Sean A , Ulrich Jason , Holtzman David M , Artyomov Maxim , Colonna Marco 
Published: January 2023

Abstract
Alzheimer’s disease (AD) is the most common form of dementia that is initiated by extracellular plaque deposition and intraneuronal hyperphosphorylated tau aggregates. Reactive astrocytosis and microgliosis are secondary cellular responses to pathology that have gained increasing attention. Variants of the microglia receptor TREM2 increase AD risk and activation of “disease-associated microglia” (DAM) is dependent on TREM2 in mouse models of AD. To investigate global transcriptomic changes during AD pathology, we surveyed gene expression changes associated with AD pathology and TREM2 in Ab-driven 5XFAD mouse model and human AD by single nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a novel Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less palpable in TREM2 R47H and R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.
Aberrant regulation of IL-1 during pregnancy promotes the development of autism-related neurodevelopmental disorders in mice
Authors: Lukens John R , Lammert Catherine Rose 
Published: January 2023

Abstract
The etiologies of most neurodevelopmental disorders, including autism spectrum disorder (ASD), remain incompletely understood. Interestingly, recent epidemiological and experimental evidence suggests that hyperactive maternal immune activation (MIA) impedes normal brain maturation and can promote the development of autism-related phenotypes. Indeed, our studies and work by others demonstrate that offspring born to pregnant mice that were exposed to the innate immune activator PolyI:C develop many of the defining behavioral features of ASD, including defects in social preference, communicative impairments, and repetitive/stereotyped behaviors. Although increasing evidence implicates key roles for dysregulated gestational inflammatory responses in neurodevelopmental disorders, the specific immune pathways that provoke autism-related phenotypes remain poorly described. In our studies, we demonstrate that inhibition of IL-1 signaling during gestation provides significant protection against the development of social abnormalities, communicative deficits, and repetitive/stereotyped behaviors in the MIA model of neurodevelopmental disease. Moreover, we find that treatment of pregnant mice with bioactive IL-1beta is sufficient to induce the development of autistic-like behaviors in the subsequent offspring. Our findings identify key roles for IL-1 in neurodevelopmental disorders and suggest that IL-1 may serve as a biomarker to identify at-risk pregnancies.
Kinetics and potency of T Cell-mediated cytolysis of glioblastoma
Authors: Clements Mike , Logun Meghan T , Chvatal Stacie , Hayes Heather B , Millard Daniel C , Karumbaiah Lohitash 
Published: January 2023

Abstract
Glioblastoma is an aggressive brain cancer with no effective treatments and a prognosis of 12–15 months. Immune system effector T cells are a promising therapy due to their high specificity and innate cytotoxicity. Assessing the efficacy and potency of T cell therapies in vitro and with high throughput is vital for development of this promising therapy. Cellular impedance-based assays offer sensitive, label-free, and continuous monitoring of cell proliferation and immune cell-mediated cytotoxicity, revealing both the potency and kinetics of T cell killing. Here, we investigated the targeting potency of activated human T cells on human U87MG glioma and patient-derived glioma cells.
U87MG and patient-derived glioma cells were plated at densities ranging from 10k to 50k cells per well on a PDL-coated CytoView-Z 96-well plate (n=12 wells per density). After 24 hours, activated patient-derived T Cells (ImmunoCult CD3/CD28 activation media) were added in a 10:1 effector:target ratio. Impedance and cytolysis were continuously monitored for over 120 hours.
As expected, higher densities of glioma cells proliferated faster and reached a higher impedance level at 24 hours. Addition of activated human T cells resulted in a decrease in impedance consistent with T cell-mediated lysis of the glioma cells. The highest density of T cells (500k) exhibited the fastest rate of U87 cytolysis, with a Kill Time 50 of 35 hours. All densities reached 100% cytolysis by 100 hours.
Overall, activated human T cells were highly effective for cytolysis of U87MG and patient-derived glioma cells. The cellular impedance assay revealed high potency, with kinetics that varied across cell densities despite the same effector:target ratios.