bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid, Large Scale Experiments to Monitor Day-to-Day Cytokine Secretion, T-Cell Activation and Immunophenotype in T-Cell Activation Protocols
Authors: Duensing Thomas , Liu Zhaoping 
Published: January 2023

Abstract
Monitoring the ex vivo activation of human T lymphocytes is key to developing optimized adoptive cell therapy protocols. We describe here the development of a large scale multiplexed assay using high throughput flow cytometry to daily monitor T-cell activation in PBMCs from multiple donors. On Day 0, PBMCs from multiple donors were profiled to confirm immunophenotype before and after T-cell enrichment with CD3/CD28 magnetic beads. Enriched T cells were cultured in the presence of the beads for 3 days. On Days 1, 2, and 3, samples were removed from the cultures and evaluated using assays that combined cell phenotype measurements with analysis of multiple secreted cytokines. In each well of the assay plates, secreted levels of 11 cytokines and multiple cellular readouts including the presence of cells expressing CD69, CD25 and HLA-DR over the course of a three-day activation protocol were determined. Cell viability and cell number for different subpopulations were also evaluated. Each 96-well plate contained T cells from 12 different donors, in quadruplicate, in two different dilutions. Each plate was read on the iQue® Screener PLUS system in less than 15 minutes. The results indicate time resolved, donor-to-donor differences in activation markers. These large-scale experiments with high throughput flow cytometry provide extensive T-cell activation profiles on multiple donors with rapid assay turnaround time. The use of these assays can provide valuable information for optimizing adoptive cell therapy development and manufacturing protocols.
A simple and integrated workflow for deep proteomic and transcriptomic analysis of sorted cell populations
Authors: Hinerfeld Douglas , Fuhrman KIt , Demirkan Gokhan , DosSantos Gary , Geiss Gary 
Published: January 2023

Abstract
In addition to the long standing efforts to understand and manipulate the immune system in the treatment of autoimmune diseases, the immune system is increasingly becoming a direct target for cancer therapeutics. Recent advances in flow and mass cytometry have greatly expanded the number of immune cell parameters that can be interrogated resulting in an improved understanding of the immune system heterogeneity. These technologies, however, remain limited in the number and types of analytes that can be examined in a single clinical sample. The NanoString nCounter® platform enables the highly multiplexed digital analysis of both RNA and protein from a single biological specimen for multiple research applications. We have recently developed the nCounter® Vantage 3D™ RNA:Protein Immune Cell Profiling Assay for research applications, which interrogates 30 cell surface proteins and 770 immune-related RNAs starting with cells in suspension. Expanding on this, we demonstrate the development of a streamlined workflow that integrates standard immune cell sorting with downstream nCounter® analysis. By co-staining PBMCs with both fluorescently-labeled and DNA barcoded antibodies, CD8+ and CD4+ T cells and CD19+ B cells were isolated followed by analysis of dozens of additional proteins and 770 RNA from each sorted population. Demonstrating the value of this workflow in analyzing potentially rare cell populations, the number of target cells were titrated to determine the sensitivity of the workflow. Without the requirement for additional molecular biology methods, such as RNA purification or sequencing library construction, this method is ideally suited for incorporation into any cell sorting workflow.
Natural killer cell development and maturation is regulated by Sostdc1
Authors: Millan Alberto Jovani , Elizaldi Sonny , Lee Eric , Murugesh Deepa , Loots Gabriela , Manilay Jennifer O 
Published: January 2023

Abstract
Natural killer (NK) cells are specialized lymphocytes with innate ability to eliminate virally infected and cancerous cells. NK cells stochastically express Ly49 activating and inhibitory surface receptors, through mechanisms that are not fully understood. Ly49 expression during NK cell development is regulated by a combination of external and internal factors, such as major histocompatibility complex class I (MHC-I) ligands and cytokines. Global deletion of sclerostin domain containing-1 (Sostdc1), a secreted protein, leads to altered bone homeostasis via dysregulation of Wnt and BMP signaling, which in turn, could result in altered immune cell development in the bone marrow (BM). Using Sostdc1-knockout (KO) mice, we have discovered a novel role for Sostdc1 in NK cell development. Flow cytometric analysis of the Ly49 repertoire in immature (CD27+CD11b-; iNK), transitional (CD27+CD11b+, tNK) and most mature (CD27-CD11b+, mNK) Sostdc1-KO NK cells demonstrated expression differences in Ly49G2, I, H, and D receptors. We also observed a progressive increase in tNK cell numbers and frequencies as the KOs aged from 12 weeks to 30 weeks, indicating a slowing or partial block in NK cell maturation with aging. Reciprocal BM chimera experiments showed that Sostdc1 regulates NK cells through cellintrinsic and cell-extrinsic manners. Consistent with this, Sostdc1 is expressed in the bone periosteum and we discovered Sostdc1 expression in CD45+ NK1.1-CD11b+ BM cells and iNK cells. Taken together, these data support a role for Sostdc1 in the regulation of NK cell differentiation and identify potential NK cell niches. Current studies are investigating whether Sostdc1-KO NK cells respond to stimulation and lyse MHC-I deficient target cells.
Diet-induced weight loss restores T cell number and function in the intestinal epithelium of obese mice
Authors: Jameson Julie M , Miranda Nadia Delgadillo , Limon Natalie , Barba Cindy , Park Christa S 
Published: January 2023

Abstract
Over 30% of adults have a BMI greater than 30 in the United States. Obesity is associated with complications such as type 2 diabetes and chronic non-healing wounds. Our laboratory has demonstrated that obesity and type 2 diabetes negatively impact T cell number, proliferation and cytokine production in the epithelial layer of the skin and the intestine. However, little is known about whether the number and function of these T cells can be restored to normal levels. Here we investigated whether diet-induced weight loss would restore T cells in the small intestine of obese mice. Mice were placed on either 10% kcal Normal Chow Diet (NCD) for 14 weeks, 60% kcal high fat diet (HFD) for 14 weeks, or 60% kcal HFD for 7 weeks followed by a 10% kcal diet for the remaining 7 weeks. The small intestine was examined by snap freezing the tissue, cutting 5um sections, staining the tissue sections for T cells, and examining the sections by immunofluorescent microscopy. Diet-induced weight loss restored T cell numbers to their original levels in the small intestine. In addition, short-term dieting restored the expression of adhesion molecules such as CD103 and chemokine receptors such as CCR9 on the IEL. Previously we have shown that obese mice are more sensitive to DSS-induced colitis. Diet-induced weight loss rescued mice from excessive bleeding and weight loss due to DSS-induced damage. Together this data suggests that while obesity impairs lymphocytes in barrier tissues such as the intestinal epithelium, diet-induced weight loss can restore T cell number and improve the outcome from colitis.
Young serum-derived exosomes potentially attenuate inflammaging via partially rejuvenating immuotolerance (Supported by NIH/NIAID R01AI121147 to D-M. S.)
Authors: Wang Weikan , Wang Liefeng , Oh Jiyoung , Zhuge Qichuan , Su Dongming 
Published: January 2023

Abstract
Aging results in a chronic inflammatory condition, termed inflammaging, establishing a risk for age-related, such as neurocardiovascular, diseases. Therefore, it is of great importance to develop rejuvenating strategies able to attenuate inflammaging as a means to help intervention of age-related diseases. A promising “rejuvenation factor” present in young blood has been found to be able to make aged neuron “younger”. However, the component in the young serum is largely unclear. Herein, we tested rejuvenation in naturally-aged mice with a potential factor – young serum-derived exosomes, based on different spectrums of microRNAs in the young and old exosomes. We found that young murine serum-derived exosomes were able to attenuate inflammaging in both the periphery and the central nerve system of old mice. In addition to senescent cells secreting pro-inflammatory factors, thymic aging-released self-reactive T cell-induced autoimmune predisposition is another potential etiology of inflammaging, we identified that the young serum-derived exosome-attenuating inflammaging was partially attributed to rejuvenation of thymic aging, characterized by reversed thymic involution and improved function in negative selection. Our pilot work provides a clue of the rejuvenation factor in the young serum, which holds great promise toward the development of novel therapeutics to reduce morbidity and mortality caused by age-related diseases.
Reactive oxygen species regulate the inflammatory function of NKT cells through Promyelocytic Leukaemia Zinc Finger (PLZF) protein
Authors: Kim Yeunghyen , Pyaram Kalyani , Kumar Ajay , Chang Cheong-Hee 
Published: January 2023

Abstract
Cellular metabolism controlled by the signaling pathways has a crucial role in dictating the outcome of T cell activation and effector function. Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as a second messenger. In T cells, ROS levels increase upon activation and antigen specific responses of T cells are compromised in the absence of ROS production by mitochondria. However, it is not well understood how NKT cells’ functions are controlled by cell metabolism. In the current study, we investigated the role of ROS in NKT cell function. We found that ROS levels were similar among CD4, CD8 and NKT cell subsets in the thymus but NKT cells showed dramatically increased ROS in the spleen and liver but not in adipose tissues. ROS in the peripheral NKT cells were primarily produced by NADPH oxidases not mitochondria. Accordingly, ROS high NKT cells were susceptible to oxidative stress and underwent apoptotic cell death. Furthermore, ROS play an important role regulating the inflammatory function of NKT cells because antioxidant treatment of NKT cells showed reduced frequencies of IFN-γ+ and IL-17+ cells. In line with this, freshly isolated ROS-high NKT cells had more NKT1 and NKT17 cells but less NKT2 than ROS-low cells. Importantly, these characteristics of NKT cells are regulated by PLZF (Promyelocytic Leukaemia Zinc Finger) protein as evidenced by reduced ROS in NKT cells from adipose tissues that do not express PLZF and also those from PLZF haplodeficient mice. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Together, our study revealed for the first time that ROS regulate NKT cell functions through PLZF.
Complement component C1q regulates AMPK and ERK1/2 activation in mouse and human macrophages
Authors: Willmann Emily A , Pandurovic Vesna , Bohlson Suzanne 
Published: January 2023

Abstract
C1q is the recognition component of the classical complement pathway and a soluble pattern recognition receptor that regulates myeloid cell activation independent of the complement cascades. C1q enhances phagocytosis of antibody opsonized targets and apoptotic cells, and regulates cytokine production in macrophages. C1q deficiency results in autoimmunity, consistent with its function in regulating macrophage activation. The signaling mechanisms leading to C1q-dependent macrophage activation are not fully understood. To investigate the mechanism by which C1q regulates macrophage activation, we utilized a phospho-kinase array to identify targets within the signaling pathway(s). The array identified several targets that were regulated by C1q including AMP-dependent protein kinase α-1 (AMPK) and ERK1/2. Short term stimulation (30 min) with C1q and subsequent addition of immune complexes resulted in enhanced ERK phosphorylation in C1q-treated THP-1 cells and mouse bone marrow derived macrophages (BMDM). These same conditions stimulate enhanced phagocytosis of targets suboptimally opsonized with IgG. Long term stimulation (18 hr) of BMDM with C1q under conditions that lead to enhanced engulfment of apoptotic cells and dampening of proinflammatory TNFα production led to enhanced AMPK activation and inhibition of ERK, consistent with C1q enhancing efferocytosis and dampening TNF production. Silencing AMPK resulted in inhibition of C1q-dependent efferocytosis, however AICAR, the AMPK agonist, was not sufficient for restoring C1q-dependent signaling. These data identify new targets of C1q-dependent signaling which may be important in developing strategies to prevent autoimmune and/or inflammatory diseases.
A tissue-resident macrophage specific coinhibitory molecule promotes regulatory T cell differentiation and stability
Authors: Fu Wenxian , Yuan Xiaomei , Yang Bi-Huei , Dong Yi 
Published: January 2023

Abstract
CD4+Foxp3+ regulatory T (Treg) cells play a central role in the prevention of lethal autoimmunity and excessive inflammation. In contrast to their counterparts in lymphoid organs, Treg cells in non-lymphoid tissues acquire tissue-specific functional properties. While Treg tissue-tropisms are increasingly recognized, it remains poorly understood how tissue microenvironment influences Treg development, function and stability. Complement receptor of immunoglobulin family (CRIg) is a recently identified B7/CD28 family member. CRIg is exclusively expressed in tissue-resident macrophages. Earlier studies have found that CRIg functions as a coinhibitory molecule to suppress T cell proliferation and cytokine production. We here report a completely novel mechanism by which CRIg regulates T cell biology. We find that CRIg promotes the development of TGF-b induced regulatory T (iTreg) cells. More importantly, CRIg stabilizes the expression of Foxp3 in Treg cells by enhancing their responsiveness to IL-2. In vivo modulation of CRIg increases Treg abundancy and restores immune tolerance in autoimmune condition. In summary, these data shed new light on how tissue-resident macrophages impact tissue homeostasis by regulating the development and stability of Treg cells. CRIg may represent a tissue-specific immunomodulation to promote immune tolerance.
ICOS signaling suppresses an excessive T cell response in the CNS during chronic T. gondii infection
Authors: O’Brien Carleigh , Harris Tajie H 
Published: January 2023

Abstract
While the importance of Tregs in limiting T cell responses during the generation of an effector immune response to pathogens has been shown, the exact mechanisms by which Tregs limit immunopathogenic T cell responses during chronic inflammation of the brain remain unknown. We find that Tregs are recruited to the brain during toxoplasmic encephalitis, where a strong effector T cell response is necessary to limit replication of reactivated parasite. Inducible T cell costimulator (ICOS) has been shown to be both critical for developing optimal effector T cell responses to pathogens, as well as necessary for Treg survival in a homeostatic state. However, the role of ICOS signaling during the maintenance of long-term immune responses to chronic inflammation remains poorly understood. We find that Tregs recruited to the CNS during chronic infection express higher levels of ICOS than effector T cells, and that blocking ICOS signaling during chronic infection unexpectedly leads to increased T cell responses and immune-mediated pathology in the brain. This immunopathogenic effect is limited to the CNS, where the increased effector T cell response seen with ICOS-ligand blockade is associated with higher CD25 and Bcl-2 expression, suggesting a lack of ICOS signaling enhances effector T cell survival in the brain during chronic inflammation. Surprisingly, no changes in the costimulatory properties of APCs in the brain have been observed, suggesting a lack of regulation within the T cell population without ICOS costimulation at sites of inflammation. The excessive T cell response seen in the absence of ICOS signaling suggests an important role for ICOS-ICOSL costimulation in maintaining local immune suppression in the CNS during chronic infection.
MicroRNAs of the miR-23~27~24 family coordinately regulate T follicular helper cell responses during allergic reaction and viral infection
Authors: Wu Cheng-Jang , Cho Sunglim , Cruz Leilani Ong , Lin Ling-Li , Chen Mei-Chi , Lu Li-Fan 
Published: January 2023

Abstract
T Follicular helper (Tfh) cells are essential for forming geminal centers (GCs) and promoting protective humoral immunity. On the other hand, aberrant Tfh cell responses could also lead to the production of autoantibodies and the development of autoimmunity. To date, microRNAs (miRNAs) have emerged as key molecular players in regulating Tfh cell differentiation and homeostasis. Here, by using both lost-of-function and gain-of-function approaches, we show that loss of miR-23~27~24 clusters in T cells resulted in elevated frequencies of Tfh and GC B cells upon allergen challenge as well as during viral infection whereas T cell-specific overexpression of this miRNA family led to reduced Tfh cell responses. Interestingly, unlike our previously reported findings which showed that individual members of this miRNA family do not always work with each other in regulating other T cell lineages, we found that the entire miR-23~27~24 family coordinately control Tfh cell responses through targeting a network of genes that are critical for Tfh cell differentiation and function. Collectively, our results establish miR-23~27~24 clusters as important regulators in maintaining optimal Tfh cell responses and the resultant humoral immunity.
Identification of a role for Casz1 in the control of T helper differentiation and inflammation
Authors: Pandiyan Pushpa , Bhaskaran Natarajan , Thiele Carol J , Weinberg Aaron 
Published: January 2023

Abstract
While T helper (Th) cells constitute a critical arm of adaptive immune system and are important for host defense, unregulated expansion and imbalance in Th effector subsets contribute to inflammatory disorders. Here we show that Casz1, whose function is previously unknown in CD4+ T cells, determines the balance between various Th subsets. As systemic knockout mice are embryonically lethal, we generated conditional CD4 Casz1 knockout mice. Conditional deletion of Casz1 in CD4+ T cells significantly inhibits Th17 cell differentiation, but promotes Th1 differentiation. Loss of Casz1 in CD4+ T cells lowers susceptibility to experimental autoimmune encephalomyelitis, consistent with the reduced frequency of Th17 cells, despite an increase in Th1 cells. These results underscore the critical role of Casz1 in determining Th17 lineage differentiation in vivo. Transcriptome analyses of Casz1 deficient CD4+ T cells show a signature consistent with defective Th17 differentiation but enhanced Th1 differentiation. Taken together, these data reveal Casz1 as a new T helper cell regulator having important clinical implications for autoimmune inflammation.
O-GlcNAc Regulates CD4+ T Cell Differentiation
Authors: Machacek Miranda E , Li Jibiao , Li Tiangang , Slawson Chad , Fields Patrick 
Published: January 2023

Abstract
Chronic inflammation is a feature of obesity and enhances the risk of heart disease, cancer, and diabetes. Specifically, pro-inflammatory TH1 and TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification (PTM) of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell differentiation. Since production of O-GlcNAc involves input from multiple metabolic pathways, the PTM acts as a nutrient sensor. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells in mice fed a high fat diet. CD4+ T cells from obese mice have elevated O-GlcNAc levels and are primed to secrete more IL-17A, the eponymous TH17 cytokine, and IFNγ, the signature TH1 cytokine. We also cultured mouse splenic CD4+ T cells with Thiamet-G (TMG), a selective inhibitor of the enzyme that removes O-GlcNAc. We find a significant increase in protein and transcript levels of IL-17A and IFNγ in TMG treated cells. Acetyl CoA carboxylase (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt, the transcription factor defining the TH17 lineage. We find that ACC1 is O-GlcNAcylated in CD4+ T cells. Collectively, our data suggest that elevated O-GlcNAc levels prime TH17 CD4+ T cell differentiation by altering ACC1 activity. Further study into how O-GlcNAcylation promotes pro-inflammatory T cell differentiation will provide insight into how nutritional excess exacerbates inflammation.
Exogenous truncated IK protein ameliorates inflammatory arthritis.
Authors: Jung SeoYeon , Choi Seulgi , Park Hye-Lim , Nam Jae-Hwan 
Published: January 2023

Abstract
The truncated IK (tIK) cytokine is known for its ability to downregulate the major histocompatibility complex class II on antigen-presenting cells and to regulate immune activation in inflammatory diseases by suppressing differentiation of pathogenic T cells and activation of macrophage. Because of these immune regulatory functions, previous research has proposed tIK as a new candidate for autoimmune disease therapy such as that for rheumatoid arthritis (RA). However, its immunological mechanism is still not identified clearly. In this study, we showed that exogenous tIK protein produced by the insect expression system suppressed the differentiation of TH1 and TH17 cells, which are key factors of inflammatory arthritis. We also showed that tIK protein reduced symptoms of the disease in the collagen antibody-induced arthritis (CAIA) mouse model. In addition, to investigate the receptor and signal transducers that directly interact with tIK and/or the intracellular molecules as a signal pathway, we performed a protein–protein interaction array. According to protein array data, FAM104B, CUTA, GSN, YEATS4 and some other proteins might interact with tIK protein. Among them some proteins may serve as the negative regulators of NF-kappaB transcription factor. Therefore, we suggest that tIK protein has the potential to act as a new therapeutic agent for RA patients, because it has a different mode of action compared with the currently-used biologics for RA, such as monoclonal antibody drugs.