bluuebunny/crossref_metadata_2025_split huggingface dataset:
Foxp3 domain-swap interface is required to suppress T helper type 2 transcriptional program in Regulatory T cells.
Authors: Van Gool Frederic , Nguyen Michelle L. , Mumbach Maxwell R. , Satpathy Ansuman T. , Anderson Mark S. , Marson Alexander , Chang Howard Y. , Bluestone Jeffrey A. 
Published: January 2023

Abstract
Regulatory T cells (Treg) play a fundamental role in maintaining immune tolerance. These cells are defined and characterized by expression of the master regulator Foxp3, a transcription factor that is crucial for Treg function and homeostasis. Natural occurring mutations in Foxp3 gene have been shown to be responsible for the immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, which is characterized by widespread autoimmune diseases. We recently identified a Foxp3 mutation in a boy that manifested a severe IPEX syndrome. This mutation, resulting in the substitution of methionine for isoleucine at amino acid 370 was determined to be part of a class of Foxp3 mutations that disrupt the dimerization motif in the domain swap interface of the protein. We have shown that, although dimerization mutants retain DNA binding, allows Treg development and certain Treg characteristics, it inevitably led to an IPEX syndrome in humans and mice. Genomic analysis of M370I mutant Treg by RNAseq, ChIPseq and HiChIP revealed a de-repression of the T helper type 2 (Th2) transcriptional program leading to the generation of Th2-like Treg. We have shown that, dimerization mutant proteins bind to multiple locations in the Th2 locus leading to increased intrachromosomal interactions and Th2 cytokines production. As expected, M370I Tregs were unable to suppress Th2 immune responses. Transgenic mice expressing M370I Foxp3 developed autoimmune diseases characterized by systemic Th2 cytokines production and skin and lung inflammation. In conclusion, disruption of the domain swap interface of Foxp3 by M370I mutation led to the development of Th2-like Treg which are functionally unable to suppress the development/function of Th2 cells.
Protein Kinase 2 (CK2) is Required for CD4+ T Cell Function in the Pathogenesis of Colitis
Authors: Yang Wei , Gibson Sara , Yan Zhaoqi , Qin Hongwei , Benveniste Etty 
Published: January 2023

Abstract
Crohn’s disease (CD) is one of the major forms of inflammatory bowel disease (IBD), characterized by chronic inflammation of the gastrointestinal tract. CD is associated with aberrant Th1 and Th17 responses accompanied by high levels of IFN-γ and IL-17, respectively. Protein kinase CK2 is a highly conserved serine-threonine kinase that is involved in several signal transduction pathways, including the JAK/STAT, NF-κB, PI3K/Akt/mTOR and Wnt pathways. Inhibition of CK2 kinase activity by small molecule inhibitor (CX-4945) or genetic deletion of CK2α in CD4+ T cells shifts the balance between Th17 and Tregs. However, the function of CK2 in the pathogenesis of colitis is unknown. Here, we demonstrate that CD4+ T cells from CK2αfl/fl dLck-Cre mice failed to induce wasting disease and intestinal inflammation, accompanied by lower levels of inflammatory cytokines and chemokines in the serum, and decreased IL-17A+, IFN-γ+ and IL-17A+/IFN-γ+ CD4+ T cells in the spleen and colon. Furthermore, CK2α controls CD4+ T cell accumulation in the spleen and colon through a cell-intrinsic mechanism. Thus, our data suggest that CK2α contributes to the pathogenesis of colitis, and that inhibition of CK2 kinase activity may be a novel therapeutic treatment for patients with CD.
Regulation of beta1 Integrin Recycling, Cell Migration and Focal Adhesion Turnover by Cell Adhesion molecule- CD13
Authors: Ghosh Mallika , Lo Robin , Devarakonda Charan , Aguilera Brian , Ivic Ivan , Shapiro Linda H. 
Published: January 2023

Abstract
CD13 is a multifunctional cell adhesion molecule that is expressed on a variety of cells where we have shown that it modulates receptor mediated endocytosis and ligand internalization to control downstream signaling pathways. In fibroblasts and epithelial cells, CD13 is localized with β1- integrin at focal adhesions (FAs), the sites of communication between the extracellular matrix and the actin cytoskeleton, prompting our exploration of potential contributions of CD13 in focal adhesion turnover, integrin endocytosis and trafficking. Phenotypically, FAs in CD13KO fibroblasts are elongated and irregular with displaced FA accessory proteins, markedly reduced actin stress fibers and fewer microtubule extensions, consistent with a link between CD13 and the control of FA dynamics. In wild type cells, CD13 and β1-integrin co internalize, traffic to Rab5+ early endosomes and recycle to the plasma membrane via Rab11a+ recycling endosomes. Pulse-chase assays with wild type and CD13 phospho tyrosine mutant confirmed that tyrosine phosphorylated CD13 must associate with the active ARF6 for β1-integrin recycling to the membrane to occur. Conversely, the absence of CD13 results in trafficking of internalized β1-integrin from early endosomes to Rab7+ late endosomes/lysosomes and a failure to return to the cell surface. Functionally, in migrating cells CD13 accumulates at the leading edge and co-localizes with IQGAP1 to regulate cell migration and adhesion. In conclusion, CD13 interacts with FAK-Src to control proper localization of focal adhesion proteins regulating focal adhesion turnover and associates with active ARF6-IQGAP1 to promote b1 integrin recycling and cell motility.
Epigallocatechin-3-Gallate suppresses neutrophil accumulation and the IL-1 beta pathway in a transgenic zebrafish model of inflammation
Authors: Nguyen Thao , Payan Brittany A. , Bondesson Maria , Mohan Chandra 
Published: January 2023

Abstract

Background
The zebrafish represents the simplest and most convenient vertebrate model for undertaking large screens because of its small size, rapid reproduction time and transparency of the body. Here, we adopt a well-established model of inflammation in zebrafish to assess the impact of Epigallocatechin-3-Gallate (EGCG), the main catechin in green tea.

Method
Tg (MPX:GFP) or Tg (MPX:mCHERRY) transgenic zebrafish were used, bearing GFP- or mCherry-tagged neutrophils, respectively. 4dpf embryos were anesthetized, and inflammation was induced by sterile tailfin-resection. The inflamed groups were separated into those that were non-treated or exposed to EGCG from 0 – 600 uM for 6 hours. 10 fish per group were then mounted alive in methyl cellulose for wide-field fluorescence imaging and analysis was done using ImageJ. Animation of neutrophil migration or accumulation was recorded using an inverted fluorescence confocal microscope till 6-hours after resection. To examine the mechanistic basis of EGCG’s effect, 37 zebrafish inflammatory genes were studied using qPCR.

Results
The static accumulation of neutrophils at the resection site was highly elevated after wounding (n=32, p&lt;0.0001), and this accumulation was reduced significantly by 31% and 43%, respectively, after exposure to EGCG 300 uM (p&lt;0.05) and 600 uM (p&lt;0.01). Dynamic neutrophil tracking studies are in progress. qPCR studies revealed 6 signature genes in the IL-1 beta pathway to be significantly down-regulated at least 2-fold after EGCG treatment.

Conclusion
EGCG suppresses inflammation in zebrafish through similar biochemical pathways as in other species. This novel animal model allows facile screening of inflammatory modulators in real-time.
Engineering antigen-specific T cells from human pluripotent stem cells
Authors: Vo Linda Thuy , Nguyen David H. , Roth Theodore , Marson Alexander , Daley George Q. , Bluestone Jeffrey A. 
Published: January 2023

Abstract
Immunotherapy has rapidly emerged as a promising treatment for many different types of cancer. Ongoing clinical trials continue to validate the efficacy of T cell-based cancer immunotherapy, such as chimeric antigen receptor (CAR) T cells, as a valid therapeutic approach. As such, new sources of both autologous and “off the shelf” T cells available in large qualities would be invaluable to widespread application of these treatments. Human induced pluripotent stem cells (iPSCs) represent a potentially inexhaustible source of clinically useful cell types. Previous studies have demonstrated the feasibility of generating T cells from iPSCs, but yields have been low due to the inefficiency of lymphoid differentiation. Here, we report a cell engineering platform capable of generating large numbers of T cells from iPSCs using a combination of transcription factors and chromatin modifiers. We initially performed a loss-of-function screen to identify regulators that enhance lymphoid potential in iPSC-derived hematopoietic progenitors. We found that repression of the Polycomb group protein EZH1 uniquely enhanced multilineage hematopoietic output from human iPSCs in vitro and in vivo. EZH1 directly modulates the expression of HSC and lymphoid genes in iPSC-derived hematopoietic progenitors and its knockdown enhances chromatin accessibility of these loci. Coupled with CAR and genome editing technologies, iPSC-derived CAR T cells release effector cytokines and mediate antigen-specific anti-tumor responses. Future work is aimed at improving antigen-specificity of iPSC-derived T cells. Together, this work highlights the utility of chromatin modifiers as cell engineering targets to enhance blood differentiation from human iPSCs.
Discrete roles and bifurcation of PTEN signaling and mTORC1-mediated anabolic metabolism underlie IL-7-driven B lymphopoiesis
Authors: Zeng Hu , Su Wei , Shi Hao , Chi Hongbo 
Published: January 2023

Abstract
IL-7 drives early B lymphopoiesis, but the underlying molecular circuits remain poorly understood, especially how Stat5-dependent and independent pathways contribute to this process. Combining transcriptome and proteome analyses and mouse genetic models, we show that IL-7 promotes anabolic metabolism and biosynthetic programs in pro-B cells. IL-7-mediated mTORC1 activation supported cell proliferation and metabolism in a Stat5-independent, Myc-dependent manner, but was largely dispensable for cell survival or Rag1 and Rag2 gene expression. mTORC1 was also required for Myc-driven lymphomagenesis. Importantly, PI3K and mTORC1 had discrete effects on Stat5 signaling and independently controlled B cell development. In fact, PI3K was actively suppressed by PTEN in pro-B cells to ensure proper IL-7R expression, Stat5 activation, heavy chain rearrangement and survival, suggesting the unexpected bifurcation of the classical PI3K-mTOR signaling. Altogether, our integrative analyses establish IL-7R-mTORC1-Myc and PTEN-mediated PI3K suppression as discrete signaling axes driving B cell development, with differential effects on IL-7R-Stat5 signaling.
Transgenic expression of human IL15 in NOD-scid IL2rg
null (NSG) mice enhances the development and survival of functional human NK cells
Authors: Brehm Michael A. , Aryee Ken-Edwin , Bruzenksi Lisa , Greiner Dale L. , Shultz Leonard D. , Keck James 
Published: January 2023

Abstract
Human NK cells have critical roles in tumor surveillance and in immunoregulation within the tumor microenvironment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function. Humanized mice are a powerful alternative to study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However human NK cell development and survival in humanized mice are severely limited. Based on previous studies showing the importance of human IL15 for NK cell development, we established NSG mice that constitutively expresses human IL15 (7.1 ± 0.3 pg/ml). To evaluate human NK cell development, 8 to 12-week-old NSG and NSG-Tg(Hu-IL15) mice were irradiated and injected intravenously with CD34+ HSC derived from umbilical cord blood. Levels of circulating human CD45+ cells, T cells and B cells were similar between the HSC-engrafted NSG-Tg(Hu-IL15) and NSG mice. Significantly higher levels of human CD56+ NK cells were found in NSG-Tg(Hu-IL15) mice as compared to NSG mice at all time points and in all tissues tested. A higher proportion of human CD56+ NK cells recovered from the blood and spleen of NSG-Tg(Hu-IL15) mice expressed granzyme A, granzyme B and perforin as compared to NK cells from NSG mice, suggesting that the NK cells were functional. Moreover, human NK cells enriched from the NSG-Tg(Hu-IL15) mice lysed K562 cells in an in vitro cytotoxicity assay. These data demonstrate that HSC-engrafted NSG mice expressing human IL15 support enhanced development of functional human NK cells and suggest that HSC-engrafted NSG-Tg(Hu-IL15) mice are a powerful model to study human NK cells.
Delineating the ILC Development Pathway
Authors: Kasal Darshan , Bendelac Albert S. 
Published: January 2023

Abstract
We investigated the hierarchical relationship of previously identified innate lymphoid cell (ILC) precursor populations in the murine bone marrow (BM) and fetal liver (FL) through the use of novel transcription factor reporter combinations and single cell potential analysis. We found that the common helper ILC progenitor (CHILP) comprises a heterogeneous population of ILC Precursors (ILCPs) and LTi precursors (LTiP), but contains no common progenitor to either as originally proposed. Remarkably, the Early ILC Progenitor (EILP), a population that is marked by the transient loss of IL-7R expression and arises prior to the ILCP and LTiP, is heterogeneous, containing both ILCP-like and LTiP-like cells with limited potential. Taken together, these results suggest that the point of helper ILC and LTi bifurcation is situated earlier than previously supposed, potentially from the EILP.
MHCII+ Macrophages in Peritoneal and Pleural Cavities Require IRF4 for Development from Circulating Monocytes
Authors: Kim Kiwook , Williams Jesse W. , Wang Ya-Ting , Ivanov Stoyan , Gilfillan Susan , Colonna Marco , Virgin Herbert W. , Gautier Emmanuel L. , Randolph Gwendalyn J. 
Published: January 2023

Abstract
Resident macrophages in peritoenal and pleural cavities exist as two distinct populations: F4/80+ICAM2+macrophages and MHCII+ macrophages. F4/80+ ICAM2+macrophages are derived from CX3CR1+embryonic precursors, and maitained by the transcription factor Gata6. However, the origin and regulating factors that maintain MHCII+ macrophages remain unknown. Here, we show that the MHCII+ macrophages arise postnatally from monocytes, which continuously replenish this subset through adulthood. Gene expression analysis identified distinct surface markers such as CD226 and revealed that the transcription factor IRF4 was selectively expressed in these macrophages relative to other organs. Monocytes first enter peritoneal or pleural cavities to become MHC II+ cells that up-regulate CD226 later as they continue to mature. In the absence of IRF4 or after administration of oral antibiotics, MHCII+CD226−CD11c− monocyte-derived cells accumulated in peritoneal and pleural cavities, but CD11c+ CD226+ macrophages were lost. Thus, MHC II+ resident peritoneal and pleural macrophages are continuously replenished by blood monocytes recruited to the peritoneal and pleural cavities constitutively, starting after birth, where they require IRF4 and signals likely derived from the microbiome to fully differentiate.
Differentiation of hematopoietic stem and progenitor cells to NK cells in a stroma-free, serum-free culture system
Authors: Tabatabaei-Zavareh Nooshin , Fawcett Kent A. , Le Fevre Tim A. , Szilvassy Stephen J. , Thomas Terry E. , Eaves Allen C. , Wognum Albertus W. 
Published: January 2023

Abstract
Natural Killer (NK) cells play an important role in innate immunity by secreting proinflammatory cytokines and killing tumor cells and virus-infected cells. Human NK cells can be generated by culturing CD34+ hematopoietic stem and progenitor cells (HSPCs) with stromal cells and cytokines. The use of stroma (and serum) is, however, not desirable in clinical applications. We developed a culture method for generating large numbers of NK cells from purified CD34+ cord blood (CB) HSPCs in the absence of serum and stromal cells. CD34+ CB cells were isolated by EasySep magnetic separation, seeded into culture plates coated with a Notch ligand and cultured for two weeks in StemSpan Serum-Free Expansion Medium (SFEM II) supplemented with SCF, TPO, Flt3L and IL-7. These conditions promote expansion of HSPCs and their differentiation into CD7+CD5+ lymphoid progenitors. Cells were then transferred to non-coated plates and cultured for two more weeks in StemSpan SFEM II supplemented with SCF, Flt3L, IL-7, IL-15 and a small molecule, UM729, to promote differentiation into NK cells. After culture, on average 76% (range: 54–90%, n=12) of cells expressed the NK cell marker CD56 and 58% (range: 25–80%) coexpressed CD56 and NKp46 (a NK cell activating receptor). A more mature CD56+CD16+ NK cell subset also emerged in these cultures (5%, range: 1–11%). The yield of CD56+ NK cells was ~13,000 (600–41,000) per initial CD34+ cell. The NK cells were functional and able to kill K562 target cells. These results show that HSPCs can expand and differentiate into NK cells under stroma- and serum-free culture conditions. This novel culture system will be useful for studies directed toward the development of cancer immunotherapies where large numbers of NK cells are needed.
Lymphoid biased hematopoietic stem cells acquire a myeloid pattern of gene expression with age.
Authors: Kong Ying , Pioli Peter D. , Montecino-Rodriguez Encarnacion , Casero David , Dorshkind Kenneth 
Published: January 2023

Abstract
Lymphocyte development declines with age, which in turn contributes to the reduced replenishment of naïve lymphocytes in secondary lymphoid organs. An impaired ability of old hematopoietic stem cells (HSCs) to generate lymphoid progeny is thought to contribute to this attenuation of lymphopoiesis. It is now recognized that the HSC compartment is heterogeneous and includes lymphoid biased (Ly-HSCs) and myeloid biased (My-HSCs) stem cells. Previous studies have demonstrated that the frequency of Ly-HSCs is reduced with age, and this observation has resulted in the formulation of a model of stem cell aging which proposes that the age-related decline in lymphocyte development is due to a reduction in the number of these lymphoid biased precursors. We now show that when total HSC number is taken into account, Ly-HSCs do not decline in number with age and that compartment size is maintained in old mice. In order to obtain insights into why lymphocyte development is reduced with age, we compared the transcriptomes of young and old Ly-HSCs. We found that 371 genes were differentially expressed between young and old Ly-HSCs, and Gene Ontology analysis revealed that multiple processes that included regulation of apoptosis, chromatin remodeling, cytoskeleton organization, and cell cycle progression were altered by aging. We also transcriptionally profiled young and old My-HSCs and analysis of the data unexpectedly revealed that the transcriptomes of old Ly-HSCs resembled that of old My-HSCs. Based on these results, we propose a revised model of HSC aging in which altered patterns of gene expression that occur in aging Ly-HSCs, rather than a reduction in their number, contribute to declines in lymphocyte production in old individuals.
Paneth cells regulate allergic sensitization in the gastrointestinal tract
Authors: Kim Eunsoo , Burnett Drew J. , Rudinsky Adam J. , Mori-Akiyama Yuko , Cormet-Boyaka Estelle , Boyaka Prosper N. 
Published: January 2023

Abstract
The gut immune homeostasis plays a major role in controlling sensitization to food allergens and the development of allergic responses in the gastrointestinal tract, but also to skin or lung. Epithelial cells sensing of commensal microbes is crucial for the maturation of the gut immune system and antibiotic treatment during early life is believed to have contributed to the increased incidence of food allergy. Paneth cells secrete antimicrobial products in the crypts of the small intestine, but their role in food allergic remains elusive. We found that SOX9ΔIEC mice, which lack of Paneth cells, display dysbiosis with increased microbial diversity and the presence of a large number of unclassified bacterial species. Upon oral sensitization with OVA as a model of food antigen in the presence of cholera toxin, SOX9ΔIEC mice developed similar levels of allergen-specific serum IgA and IgG responses to control wild-type mice with the exception of IgG3 responses, which were elevated in SOX9ΔIEC mice. Interestingly, SOX9ΔIEC mice exhibited significantly increased allergen-specific serum IgE responses, and exacerbated signs of allergic responses (i.e., swelling, hypothermia, impairment of lung functions) after nasal allergen challenge. Transplantation of fecal materials from SOX9ΔIEC mice was able to transfer the hyper-allergenic phenotype to control wild-type mice. Ongoing studies are assessing the metabolites present in the intestinal lumen of SOX9ΔIEC mice and their effect on intestinal epithelial cells and myeloid cells to elucidate the mechanisms by which impaired or defective anti-microbial functions of Paneth cells regulate allergic sensitization of the GI tract.
Development and validation of a novel method to evaluate differences in allergenicity among wheat genotypes
Authors: Gangur Venugopal , Gao Haoran , Jin Yining , Ng Perry KW. , Olson Eric 
Published: January 2023

Abstract
Wheat allergy is one of the major food allergies that is growing at an alarming rate for reasons that are not completely understood. Variation in allergenicity among various wheat genotypes is not well studied. Here we sought to develop a novel method to evaluate the relative allergenicity among wheat genotypes. Groups of Balb/c mice weaned onto a plant-free diet received four IP injections with AABB or AABBDD wheat salt-soluble protein extract and alum. After confirming IgE responses and clinical sensitization for anaphylaxis, mice were boosted repeatedly over a 6-month period. Bi-weekly blood samples were collected and pooled to create AABB and AABBDD plasma banks. Using this plasma, IgE inhibition ELISA systems were optimized and used to determine the relative allergenicity. In the AABB system, AABBDD abolished IgE binding by 81+/−5%, and DD SSWP abolished it by 69+/−4%. In the AABBDD system, AABB abolished IgE binding by 76+/−4% and DD abolished it by 80+/−8%. In the AABB system, other genotypes were less allergenic by 7% (AABBDD) and 21% (DD). In the AABBDD system, other genotypes were less allergenic by 12% (DD) and 17% (AABB).