bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in chronic rhinosinusitis
Authors: Kato Atsushi , Poposki Julie A , Klingler Aiko I , Stevens Whitney W , Peters Anju T , Hulse Kathryn E , Grammer Leslie C , Schleimer Robert P , Welch Kevin C , Smith Stephanie S , Sidle Douglas M , Conley David B , Tan Bruce K , Kern Robert C 
Published: January 2023

Abstract
Thymic stromal lymphopoietin (TSLP) is known to be elevated and truncated in nasal polyps (NPs) of chronic rhinosinusitis and might play a significant role in type 2 inflammation in this disease. However, neither the structure nor the role of the truncated products of TSLP has been studied. We investigated the mechanisms of truncation of TSLP in NPs and the function of the truncated products. We incubated recombinant full-length 15.1 KDa human TSLP (methionine + residues 29–159, referred to as M29–159) with NP extracts. SDS-PAGE revealed two major truncated products of TSLP in NP extracts. Edman sequencing and MALDI-TOF MS results indicated that the longer truncation product was TSLP (M29–124) and the smaller product was TSLP (131–159). We found that PCSK (proprotein convertase subtilisin/kexin) family proteases were rate-limiting enzymes in the truncation and generation of TSLP (M29–130) and TSLP (131–159). Carboxypeptidases further digested 6 amino acids from the C-terminus of TSLP (M29–130) to generate TSLP (M29–124) in NPs. Interestingly, recombinant TSLP (M29–124) and TSLP (131–159) did not manifest TSLP activity, but PCSK-digested TSLP had significantly higher activity than full-length TSLP on myeloid dendritic cells and group 2 innate lymphoid cells. Finally, we identified a 14.2 KDa TSLP from incubation of TSLP with NP extracts under non-reducing conditions that was significantly reduced in the presence of DTT. This indicates that the two cleaved products are dimerized with disulfide bonds and the dimerized form, TSLP (29–124 + 131–159), is an active metabolite and may contribute to the amplification of type 2 inflammation in NPs.
MiR-205 Supports Thymopoiesis Following Stress by Positively Regulating Foxn1 Expression
Authors: van Oers Nicolai S , Hoover Ashley R , MacLeod Jessica , Dozmorov Igor , de la Morena M. Teresa 
Published: January 2023

Abstract
T cells are a critical component of the adaptive immune system, developing within the thymus. Immature thymocytes interact with an interconnected meshwork of thymic epithelial cells (TECs) to establish the T cell repertoire. The developmental process is extremely stress sensitive, as the thymus can undergo a rapid involution in response to diverse inflammatory conditions, and a more prolonged hypoplasia during aging. The type of stress predicates whether TECs, thymocytes, and/or myeloid cell populations are affected. Several microRNAs (miRs) have been identified in the thymus based on their ability to mitigate the stress damage. We identified miR-205 as a stress responsive miR specifically expressed in TECs. Mice generated with a conditional ablation of miR-205 in TECs exhibit an age and sex-dependent thymic hypoplasia beginning at 8 weeks. Under stress conditions involving a type I interferon response (dsRNA mimic; polyI:C), the TEC-miR-205 deficient mice had a severe thymic atrophy compared to littermate controls. The TEC-miR-205 deficient mice displayed a delayed recovery of single positive CD4 and CD8 thymocytes, likely resulting from a block in the cortical TEC expansion. qPCR and gene expression comparisons revealed that the miR-205 deficient TECs had significant changes in chemokine/chemokine receptor and antigen processing pathways. MiR-205 positively regulated Foxn1 transcription factor expression, the master regulator of TEC development and function. Interestingly, miR-205 is encoded within a long non-coding RNA (lncRNA), 4631405K08Rik. Current experiments will reveal the mechanisms by which the miR and lncRNA are transcriptionally regulated, and how these affect Foxn1 expression to support thymopoiesis.
Alteration of TCR Dβ sequence content alters Treg development.
Authors: Levinson Michael , Silva-Sanchez Aaron , Sleckman Barry P , Zhuang Yingxin , Schroeder Harry W 
Published: January 2023

Abstract
Diversity of the T cell receptor is mainly developed through V(D)J rearrangement and N addition. The product of this rearrangement is the CDRβ3, a region of high variability that recognizes peptide and includes all of the D gene. Interestingly, the Dβ sequence is highly conserved in animals that use an adaptive immune response. This suggests that there are naturally selected germline encoded constraints on the TCR; we hypothesize that these constraints are there to streamline the process of T cell generation by biasing the repertoire into one that can recognize the most amount of antigens while limiting deleterious T cells from reaching the periphery. Because of this conservation, we hypothesize that altering the D region will have an effect on the development of thymocytes and function of mature T cells.
To test this, we created Dβ altered mice: a replacement of the Dβ locus with a charged Dβ (DβDKRQ) and a replacement of the Dβ locus with a hydrophobic DH (DβYTL). Compared to WT, the mutant mice have an altered TCR repertoire in both CDRβ3 amino acid composition and length; these differences can be attributed to the changes in the germline sequence. Changing the Dβ also changes T cell numbers in developing and mature T cells, with an altered Dβ being selected against. We also found functional differences; there is decreased epitope recognition in the DβYTL mice after immunization with ovalbumin. These data supports our hypothesis that alteration from the preferred sequence will have effects on T cell development and function.
UTX controls lineage-specific epigenetic program and development of iNKT cells
Authors: Beyaz Semir 
Published: January 2023

Abstract
iNKT cells are innate-like lymphocytes that protect against infection, autoimmune disease, and cancer. However, the role of epigenetic regulation in iNKT cell biology remains elusive. Here, we show that the H3K27me3 histone demethylase UTX is an essential cell-intrinsic factor for iNKT cell development in the thymus. UTX-deficient iNKT cells exhibit impaired expression of iNKT signature genes, including NK cell receptors and the transcription factor Tbx21. Epigenetic profiling uncovers a decrease in activation-associated H3K4me3 and an increase in repressive H3K27me3 marks within the promoters of downregulated signature genes. UTX binds to these promoters and its enzymatic demethylase activity is important for establishing the iNKT gene expression program. Moreover, we identify the AP-1 transcription factor JunB as novel master regulator of the iNKT cell gene signature, highlighted by its molecular interaction with UTX. Finally, iNKT cells harbor super-enhancers for their key factors T-bet, PLZF, and JunB, underlining their role in lineage specification.
Conditional disruption of Raptor reveals an essential role for mTORC1 in B cell development, survival, and metabolism.
Authors: Iwata Terri , Ramirez Julita , Margineantu Daciana , Hockenbery David , Tsang Mark , Iritani Brian M 
Published: January 2023

Abstract
Mechanistic target of Rapamycin (mTOR) senses diverse environmental conditions including nutrient, growth factor and stress conditions, to regulate cellular growth, division, and differentiation. Studies examining the role of mTOR signaling in immune function have revealed critical roles for mTOR in regulating T-cell differentiation and function. However, there have been few studies investigating the role of mTOR in early B cell development and function. We found that mTORC1 activity is highly activated during the pro- and pre-B stages of mouse B cell development. Disruption of mTORC1 signaling in developing mouse B-cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B-cells and loss of antigen-specific antibody production. Pre-B cell survival and proliferation were also significantly reduced in B-cells from mTORC1-signaling deficient mice. mTORC1-signaling deficiency resulted in a significant decrease in both oxidative phosphorylation and glycolysis indicating that loss of mTORC1 signaling in B cells results in altered cellular metabolism. Forced expression of a transgenic B-cell receptor on mTORC1-signaling deficient B-cells failed to rescue the developmental block. Collectively, our data reveal a previously uncharacterized role of mTORC1 signaling in early B-cell development and metabolism.
SRT1720 inhibition of M1 macrophages and chronic inflammation in Chagas disease
Authors: Wan Xianxiu , Koo Sue-Jie , Wen Jianjun 
Published: January 2023

Abstract
Trypanosoma cruzi (T. cruzi) causes Chagas disease that is one of the most frequent causes of heart failure and sudden death in Latin America. We observed the therapeutic effect of SRT1720 and explored its immunological mechanisms especially its effect on macrophage phenotype and function in chronic Chagasic mouse. SRT1720, an SIRT1 agonist, was injected intraperitoneally in T. cruzi infected mice at 1 mg/mouse/dose during day 45–66 post-infection (pi, three times a week). Mice were harvested during chronic disease phase (~150 days pi). Left ventricular dysfunction was prevented in SIRT1720-treated chagasic mice. SRT1720 ameliorated the clinical severity of chronic Chagas diseases, decreased proinflammatory cytokine (IFN-γ, IL-1 β, TNF-α) gene expression and inflammatory infiltrate in chronic chagasic hearts. The expression of iNOS, a specific marker for M1, was decreased in SIRT1720-treated chagasic heart. Splenic evaluation by flow cytometry showed both M1 (CD80+ and CD64+) and M2 (CD200R+ and CD206+) macrophages were activated in chronically-infected mice, and SRT1720 inhibited T. cruzi -induced increase in M1 macrophages. These results demonstrated that SRT1720 has therapeutic potential in Chagas disease possibly through preventing the polarization of M1 macrophages and inhibiting inflammatory responses. In vitro experiments will be done to confirm these results. Spleen macrophages will be isolated from these mice and will be cultured for 24h. The release of NO and accumulation of iNOS and secretion of IFN-γ, IL-1 β, TNF-α in cultured macrophages will be examined.
IL-4 Rescues Surface IgM Expression in Chronic Lymphocytic Leukemia
Authors: Guo Benchang , zhang Lu , Chiorazzi Nicholas , Rothstein Thomas L 
Published: January 2023

Abstract
Chronic lymphocytic leukemia (CLL) cells express poor levels of surface Ig, and many are minimally activated or anergic in response to BCR crosslinking in vitro. Paradoxically, CLL cells in patients are highly activated through BCR signaling and expand in proliferation centers, suggesting that the function of sIg signaling is rescued. Here, we find that, compared with normal naïve B cells, CLL cells express an extremely low level of CD79b protein but relatively normal levels of CD79a and IgM protein. Association of both CD79a and CD79b to IgM is markedly reduced. We further find that IL-4 markedly rescues CD79b and sIgM protein levels in CLL cells. These changes significantly enhance signaling in response to BCR crosslinking. Furthermore, we find that these changes are more pronounced in IGHV-unmutated CLL than IGHV-mutated CLL cells. The results described herein reveal that reduced sIgM is due to low expression of CD79b protein in CLL cells. Crosstalk between the IL-4 receptor and the BCR rescues sIgM-BCR expression and signaling. The differential responses of IGHV-unmutated and -mutated CLL cells to IL-4 may reflect, or be involved in, the distinct clinical courses that characterize these populations.
Foxp3-driven miR-23~27~24 clusters control regulatory T cell-mediated immune regulation
Authors: Wu Cheng-Jang , Cho Sunglim , Cruz Leilani Ong , Lin Ling-Li , Lu Li-Fan 
Published: January 2023

Abstract
CD4+ Foxp3+ regulatory T (Treg) cells maintain immune homeostasis by regulating pathologic immune responses against self-, innocuous as well as dangerous foreign antigens. Previously, microRNA (miRNA)-mediated gene regulation has been shown to play a pivotal role in the maintenance and function of Treg cells. Here, we report that miR-23~27~24 clusters, which are abundantly expressed in Treg cells, are critical in controlling Treg cell biology. Foxp3 directly promotes elevated expressions of miR-23~27~24 clusters required for optimal Treg cell function through binding to the “enhancer element” at the miR-23~27~24 loci. Loss of miR-23~27~24 clusters in Treg cells lead to the development of spontaneous lympho-hyperactivation phenotypes over time. Moreover, upon allergen challenge, mice with Treg cell-specific ablation of miR-23~27~24 clusters exhibited elevated eosinophilic airway inflammation. Together, our results identify a Foxp3-driven miRNA family that plays important roles in controlling Treg cell-mediated immune regulation at both physiological and pathological settings.
Image based machine learning for identification of M1 and M2 macrophages
Authors: Rostam Hassan Muhammad , Reynolds Paul M. , Singh Sonali , Alexander Morgan R , Gadegaard Nikolaj , Ghaemmaghami Amir 
Published: January 2023

Abstract
Macrophages play a crucial role in orchestrating immune responses against foreign materials, pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at the two ends of the spectrum. Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time-consuming, require large numbers of cells and are resource intensive. In this study, we have used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Macrophages were polarised to M1 and M2 phenotypes by GM-CSF+IFN-gamma and M-CSF+IL-4, respectively. Control cells were un-polarised (naïve) macrophages and monocytes. The efficiency of macrophage polarisation was confirmed by quantifying M1 and M2 specific surface markers, cytokine profile and transcription factors. Fluorescent microscopy was used to assess cell morphology of all cell types which stained only for nucleus and actin distribution using DAPI and phalloidin, respectively. Using only the analysis of the morphological features we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes. We were able to classify all 5 cell types with an average accuracy of 70%, whilst concentrating on classification of M1/M2 cells with a binary classifier was, on average, correct 88% of the time. Thus we suggest high content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers.
Controlling macrophage phagocytosis towards healthy self cells: Two layers of activating and inhibitory mechanisms
Authors: Bian Zhen , Shi Lei , Lv Zhiyuan , Tremblay Alexandra V , Niu Shuo , Zen Ke , Zhang Yongliang , Liu Yuan 
Published: January 2023

Abstract
Rapid clearance of adoptively transferred CD47-null cells in mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of ‘self’, and its interaction with macrophage SIRPα triggers inhibitory signaling through SIRPα cytoplasmic ITIMs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotype, the mice deficient of CD47 manifest no enhanced macrophage phagocytosis towards self, a phenomenon suggesting that additional mechanisms besides CD47-SIRPα command macrophage behavior. Studying our recently established SIRPα-knockout (SIRPα−/−) mice, as well as CD47−/− mice, we reveal that macrophage phagocytosis toward self is not only inhibited by CD47-SIRPα signaling but also requires an activation mechanism induced by unique inflammatory stimuli. SIRPα−/− mice and CD47−/− mice, though being healthy under normal condition, develop severe anemia and splenomegaly owing to splenic macrophages aggressively phagocytizing self RBC under chronic colitis, peritonitis, inflammatory cytokine treatments, or CFA and LPS-induced inflammation. Ex vivo phagocytosis assays demonstrate that macrophages from SIRPα−/− or CD47−/− mice, and even peritoneal macrophages from WT mice, are generally incompetent to attack self cells albeit their aggressiveness towards bacteria, fungi, apoptotic cells and immune complexes. However, treating all macrophages with IL-17, LPS, IL-6, IL-1β and TNFα, but not IFNg, dramatically initiates potent phagocytosis towards self cells providing an absence of the SIRPα-CD47 restraint. In addition, we identify that WT spleen red pulp is one specific environment that stimulates macrophage phagocytosis and that is lacking in deficient mice.
Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16
Authors: Srpan Katja , Cartwright Adam N. R. , Davis Daniel M 
Published: January 2023

Abstract
Natural Killer (NK) cells are cytotoxic lymphocytes that can recognize and kill virally infected or tumour transformed cells by the secretion of cytolytic granules containing perforin. An individual NK cell can kill several target cells sequentially. Each target cell can trigger NK cell activation via different activating ligands and here we report that the order in which ligands are encountered affects the NK cell response. When NK cells are repeatedly activated via their Fc receptor CD16, with the therapeutic antibody rituximab, perforin secretion decreases with each stimulation. However, perforin secretion is restored to its initial level upon subsequent activation by MICA, which ligates NKG2D. Repeated stimulation of NK cells via MICA also decreases the degranulation capacity of NK cells but, strikingly, this effect cannot be rescued by a subsequent stimulation with rituximab. We found that the mechanism underlying these differential outcomes involves shedding of CD16, which occurs upon NK cell activation through both CD16 and NKG2D. Shedding of CD16 renders the cells insensitive to further activation via that receptor but they remain competent for further activation by NKG2D. We also found that NK cells are more motile on rituximab-coated surfaces than on MICA-coated surfaces and their migration speed decreases upon inhibition of CD16 shedding. Thus, although CD16 shedding hinders NK cell responsiveness it can serve to augment NK cell motility to move between target cells. Together these data indicate that manipulation of CD16 shedding might be an important target for NK cell-based therapies including treatments with therapeutic antibodies.
TCR tuning and exhaustion of CD4+ T cells by persisting antigen
Authors: Obst Reinhard , Trefzer Anne , Pennavaria Stefanie , Wang Shu-Hung , Lober Benedikt , Asoyan Ayuna 
Published: January 2023

Abstract
Chronic infections and persistent antigen presentation by tumor cells functionally impair T cells over time. To address how different kinetics and dosage of antigen presentation affect CD4+ T cells, we followed TCR transgenic T cells transferred into antigen-transgenic recipients where they received transient or chronic TCR signals of varying strengths. In one of the recipient mouse lines, where antigen presentation can be easily regulated by doxycycline treatment, the quick termination of antigen presentation after priming allowed for the differentiation of memory cells with Ly6C, IL7R and cytokine production upon restimulation as their hallmarks. Persistent antigen presentation at three different levels led to different types of exhaustion that developed with different kinetics and consequences: Exhaustion markers and dysfunctionalities took longer to appear at low amounts and only at the highest antigen dose the T cells downregulated their TCRs and underwent apoptosis. We tested the Ca2+, MAPK and Akt pathways for functionality and found Ca2+ fluxes to be rather robust against persistent antigen presentation while the MAPK and Akt pathways were more easily tuned by it. These data correlate, for CD4+ T cells, several levels of exhaustion with antigen dosage in an otherwise sterile environment.
Basophil-derived IL-6 regulates TH17 cell differentiation and CD4 T cell immunity
Authors: Yuk Chae Min , Park Hyeung Ju , Kim Dong-Eon , Kim Tae Woo , Lee Seung-Hyo Lee 
Published: January 2023

Abstract
Basophils are rare circulating granulocytes and have been proposed to be involved in T helper (TH) type 2 immunity mainly through interleukin-4 (IL-4) secretion. In addition to IL-4, it has been known that basophils produce IL-6 and tumor necrosis factor (TNF)-α with immunoglobulin E (IgE) crosslink. TH17 cell development is achieved with the action of IL-6 and transforming growth factor (TGF)-β. Whether the capacity of basophils to produce IL-6 is involved in the differentiation of TH17 cells is unknown. The aim of this study is to determine the role of basophils on TH cell differentiation especially TH17 cell development and plasticity of TH subsets by using in vitro T cell differentiation and cholera toxin (CT)-mediated TH17 inflammation model. Bone marrow (BM) derived-basophils (BMBs) as well as splenic basophils produced large amount of IL-4 and IL-6 by stimulation with IgE crosslink or CT. BMBs have additional role of TH17 cell differentiation by supplementing the function of dendritic cells through secretion of IL-6 in vitro culture system in the presence of TGF-β. Furthermore, basophil-mediated TH17 cell differentiation effects were prominent in CT-mediated TH17 dominated lung inflammation model. Therefore, our results indicate that basophil is an important inducer of TH17 cell differentiation, which is dependent on IL-6 secretion.
An essential role for Itk in Th9 differentiation via TCR-mediated induction of IL-2 and IRF4
Authors: Gomez-Rodriguez Julio Washington , Meylan Francoise , Handon Robin , Hayes Erika , Anderson Stacie , Kirby Martha , Siegel Richard M , Schwartzberg Pamela L 
Published: January 2023

Abstract
T helper 9 (Th9) cells produce IL-9, a cytokine implicated in allergic asthma and autoimmunity. We show that Itk, a mediator of T-cell receptor signalling in Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation in vivo and in vitro. In a papain-induced model of allergic lung disease, Itk-deficient mice have reduced pulmonary inflammation and IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal induction of ILC2s. In vitro, naïve Itk−/− CD4+ T cells do not produce IL-9 and have reduced levels of IRF4, a critical transcription factor for effector T-cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or expression of constitutively-active STAT5, but not NFATc1. STAT5 binds the Irf4 promoter, indicating a mechanism by which IL-2 rescues weakly-activated T cells. Itk inhibition also reduced IL-9 expression by human CD4 T cells, thereby implicating ITK as a possible target to prevent Th9-mediated inflammatory diseases.