bluuebunny/crossref_metadata_2025_split huggingface dataset:
The phagocyte NADPH oxidase (NOX2) regulates adaptive immune response at the level of both T cells and APCs (138.7)
Authors: Williams Mark , Shatynski Kristen , Chen Haiyan 
Published: January 2023

Abstract
Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In addition to this innate immune dysfunction, CGD patients or animal models display an altered, perhaps augmented, adaptive immune response. It has been previously shown that T cells express the phagocyte oxidase and also been proposed that NOX2 expression alters APC function. The goal of this study was to determine if the differences in adaptive responses are inherent to T cells, or if other cells create an environment that promotes T helper polarization. Anti-CD3 activation of purified naive T cells from NOX2-deficient mice led to augmented IFNγ and IL-17 secretion. NOX2-deficient antigen presenting cells (APC) showed selective increases in GM-CSF, IL-6, TNF and MCP-1 induced by LPS challenge in vitro. APC function was measured using OVA-specific OT-II T cells, and oxidase deficient APC induced more IFNγ and IL-17 upon antigen stimulation. Adoptive transfer of OT-II T cells into wild type or NOX2(-/-) hosts followed by immunization with OVA in the presence of either CFA or Alum revealed altered cytokine profiles of T cells after restimulation in vitro. These results suggest roles for the phagocyte NADPH oxidase in shaping the adaptive and innate immune responses in both a T cell and APC dependent fashion.
Calcium Signaling Promotes Targeted Recycling of the Lateral Border Recycling Compartment (LBRC) During Diapedesis (140.1)
Authors: Han Fei , Florey Oliver , Feng Gong , Muller William 
Published: January 2023

Abstract
Leukocyte transendothelial migration (diapedesis, TEM) is a crucial step in both the innate and adaptive immune responses. We have shown that targeted recycling of membrane from a reticulum localized close to the endothelial cell (EC) lateral border (the lateral border recycling compartment, LBRC) surrounds the transmigrating leukocyte during TEM (Mamdouh, Z., et al 2003. Nature). Targeted recycling is required for TEM to proceed. However the mechanisms underlying recruitment of the LBRC are not entirely clear. An increase of cytosolic free calcium (Ca) in ECs and the resulting activation of Ca/Calmodulin-dependent myosin light chain kinase (MLCK) are crucial for diapedesis to occur. Here we address the role of Ca signaling in the process of targeted recycling. The cell permeant Ca chelator BAPTA-AM was loaded into ECs under conditions that blocked calcium flux in response to histamine, but did not impair cytoskeletal or junctional integrity. Under these conditions, blocking of cytosolic free Ca flux in ECs abolished targeted recycling of the LBRC, as well as leukocyte transendothelial migration to the same extent (&gt; 80%). Furthermore, blockade of MLCK with ML7 (1uM) also blocked targeted recycling and TEM by &gt; 80%. This study demonstrates that Ca signaling is upstream of LBRC targeted recycling, and MLCK activation (presumably by Ca/Calmodulin dependent mechanism) is required for targeted recruitment of the LBRC.
The S1P1 receptor antagonist W146 induces lymphopenia in mice. Demonstration that functional antagonism of S1P1 is the mechanism of lymphopenia evoked by fingolimod-like compounds. (140.15)
Authors: Godessart Nuria , Mustafa Sanam , Dolgachev Vlad , Aulí Mariona , Aguilar Núria , Cabedo Judit , Calbet Marta , Domènech Teresa , Domínguez María , López Rosa , Prats Neus , Milligan Graeme , Kunkel Steven , Tarrasón Gema 
Published: January 2023

Abstract
Agonists of sphingosine 1-phosphate receptor 1 (S1P1) are a novel class of immunosuppressants. S1P is the endogenous agonist of S1P1 and promotes lymphocyte egress from lymph nodes (LN) into blood. However, synthetic agonists like fingolimod prevent the egress, causing systemic lymphopenia. This apparent contradiction is explained by the concept of functional antagonism, according to which synthetic S1P1 agonists induce receptor internalization, making cells unresponsive to the endogenous S1P. If this mechanism is true, S1P1 antagonists should induce lymphopenia. We have fully characterized compound W146, a S1P1 antagonist, by binding and cellular assays. We then administered W146 and several S1P1 agonists to mice and measured blood lymphocytes and plasma levels of compounds in different experimental conditions. All compounds induced lymphopenia in blood and a concomitant increase of CD4+ and CD8+ T cells in LN. These results indicated that lymphopenia evoked by W146 was due to S1P1 blockade and not to another mechanism, like cytotoxicity. By Western blot, FACS and immunocytochemistry we showed that all compounds but W146 induce S1P1 internalization. W146 is able to prevent the internalization induced by an agonist. We report, for the first time, lymphopenia with a S1P1 antagonist. Plasma levels needed to surmount the effects of endogenous S1P are achieved in our experimental conditions. Our studies strongly support the validity of the concept of functional antagonism.
Endotoxin Tolerance represents a state of alternative polarization in mononuclear cells (142.4)
Authors: Pena Olga , Raj Disha , Pistolic Jelena , Fjell Christopher , Hancock Robert 
Published: January 2023

Abstract
Classical (M1) and alternative (M2) polarization of mononuclear cells such as monocyte and macrophages, mainly occurs as a response to micro-environmental challenges such as the incursion of a pathogen. Lipopolysaccharide, also known as endotoxin (LPS), is a potent inducer of inflammation and M1 polarization. LPS can also generate an effect in mononuclear cells known as endotoxin tolerance, defined as the reduced capacity of the cell to respond to LPS activation following a first exposure to this stimulus. Using systems biology approaches in peripheral blood mononuclear cells and monocyte derived macrophages, we identified that endotoxin tolerance is characterized by a similar state of alternative polarization, featuring expression of genes and proteins critical for the development of key M2 functions, including reduced production of pro-inflammatory mediators, expression of genes involved in phagocytosis as well as tissue remodeling. Moreover, expression of different methallothionein gene isoforms, known for their role in the control of oxidative stress and immunomodulation was also found consistently up-regulated during endotoxin tolerance. These results favor the hypothesis that, after an initial inflammatory stimulus, mononuclear cells undergo an alternative polarization most probably to control and balance prospective hyper-inflammation.
Increased severity of spontaneous autoimmune thyroiditis (SAT) and salivary gland infiltration in NOD.H-2h4 mice lacking regulatory T cells (T reg) (143.25)
Authors: Ellis Jason , Braley-Mullen Helen 
Published: January 2023

Abstract
NOD.H-2h4 mice given NaI in the drinking water develop SAT with chronic inflammation of the thyroid by T and B cells and production of anti-thyroglobulin (Tg) autoantibody. CD28-/- NOD.H2-4 mice, which have greatly reduced numbers of CD4+CD25+FoxP3+ Treg, were developed in order to examine the role of T reg in SAT development in WT and B cell-deficient (B-/-) mice. CD28-/- NOD.H2-h4 mice given NaI water develop more severe SAT than WT mice with collagen deposition (fibrosis) and low serum T4. Lymphocyte infiltration of salivary glands is also increased compared to WT mice. Most 4-6 mo old CD28-/- mice not given NaI water also develop SAT, whereas WT NOD.H-2h4 mice not given NaI water rarely develop SAT until much later. Although anti-Tg autoantibody levels generally correlate with SAT severity in WT mice, CD28-/- mice have reduced anti-Tg autoantibodies. Over time, the percentage of splenic follicular B cells decreases and marginal zone B cells increase. B -/- NOD.H-2h4 mice are resistant to SAT, but they develop SAT after transient depletion of T reg. CD28-/-B-/- mice, which lack most T reg, develop SAT comparable to WT NOD.H-2h4 mice. In B-/- mice, a lack of B cells as APC during the initiation phase of SAT may lead to preferential activation of Treg leading to reduced activation of effector T cells and reduced SAT severity. T reg also regulate SAT development if B cells are present since severe SAT develops in CD28-/- mice lacking T reg. (Supported by NIH RO1 AI076395)
CD137 expression distinguishes two functional CD4+CD25+ T regulatory subsets in Type one diabetes. (143.3)
Authors: Kachapati Kritika , Wu Yuehong , Mittler Robert , Wicker Linda , Ridgway William 
Published: January 2023

Abstract
The treatment of nonobese diabetic (NOD) mice with anti-CD137 monoclonal antibody prevented diabetes by increasing the number of T regulatory cells, which transferred protection from disease. We show here that two subsets of Tregs, CD137pos and CD137neg, differ phenotypically and functionally. CD137pos Tregs are distinctly CD69hi, CD62Llow and produce significantly higher levels of IL-10 and ICOS. There is significant decline in the number of CD137pos Tregs in NOD spleen but not thymus with age. This decline is not observed in an NOD.Idd9.3 congenic strain that is protected from diabetes and has a B10 Cd137 allele. The NOD.Idd9.3 strain has significantly higher numbers of CD137pos Tregs in both spleen and thymus compared to NOD mice. Functionally, NOD CD137pos Tregs are more suppressive than CD137neg cells. Here we dissect the mechanism of suppression and find that in a contact independent transwell system, the combination of anti-IL-10R and anti-TGF-β blocking antibodies decreases but does not abolish suppression of CD137pos Tregs. The loss of peripheral CD137pos Tregs with age in NOD mice, the increase in the frequency of CD137pos Tregs with anti-CD137 antibody treatment, and the persistently higher numbers of CD137pos Tregs in NOD.Idd9.3 congenic mice suggest that this functionally superior Treg subset could act to prevent diabetes when increased by genetic or environmental means. Therefore CD137pos Tregs are a therapeutic target in T1D and other autoimmune diseases.
Study of peripheral tolerance in ADA SCID patients after different treatments (143.36)
Authors: Brigida Immacolata , Sauer Aisha , Selleri Silvia , Scaramuzza Samantha , Ripamonti Anna , Casiraghi Miriam , Ferrua Francesca , Olek Sven , Roncarolo Maria Grazia , Aiuti Alessandro 
Published: January 2023

Abstract
Adenosine deaminase (ADA)-SCID is characterized by increased purine metabolites and severe combined immunodeficiency. Autoimmune manifestations have been observed in milder forms or after enzyme replacement therapy (ERT), and recently in patients following gene therapy (GT). nTregs have a full enzymatic machinery to generate and sustain high concentrations of extracellular adenosine and may be involved in the pathogenesis of autoimmunity. We analyzed the frequency of CD4+CD25+FOXP3+CD127- in the peripheral blood, the methylation profile of the FoxP3 gene and the suppressive function of nTreg in ADA-SCID patients following ERT, GT or bone marrow transplant (BMT) as compared to controls. We found that the proportion and function of nTregs after GT mirrored that of controls, while nTreg from the ERT group showed a reduced frequency and impaired function. We also investigated the maturation state of B cells in the peripheral blood of ADA-SCID patients. After ERT and early after GT, patients displayed an increased proportion of transitional B cells (CD24highCD38high) and of cells with an incomplete BCR (CD21-CD35-) as compared to BMT and healthy controls. At later follow up patients treated with GT showed a tendency to normalize the phenotype in parallel to an increased frequency of gene corrected cells. Collectively, these data provide a first indication of predisposition to autoimmunity in ADA-SCID and the role of different treatments in the maintenance of peripheral tolerance.
Evidence supporting the existence of a novel histaminergic pathway in the regulation of EAE susceptibility (143.51)
Authors: Saligrama Naresha , Del Rio Roxana , Case Laure , Noubade Rajkumar , Teuscher Cory 
Published: January 2023

Abstract
Histamine (HA) is a key regulator of experimental allergic encephalomyelitis (EAE), the autoimmune disease model of multiple sclerosis (MS). Histidine decarboxylase deficient mice (HDCKO), which are unable to synthesize HA, exhibit more severe EAE and increased IFN-γ production by splenocytes in response to MOG35-55. HA exerts its effects through four different G protein coupled receptors (GPCR): H1, H2, H3 and H4 (H1-4R). Each HA-receptor has been shown to influence EAE pathogenesis. In the mammalian brain there is, however, evidence for the existence of non-GPCR signaling by HA which is picrotoxin-sensitive and mediated by chloride conductance. GABAA receptor subunits can form HA-gated chloride channels in vitro suggesting that an ionotropic HA-receptor might contain known ligand-gated chloride channel subunits. To test the hypothesis that non-GPCR signaling by HA plays a role in immune responses, we generated H1-4RKO mice and studied their susceptibility to EAE. Here we report that in contrast to HDCKO mice, H1-4RKO mice develop less severe EAE compared to WT animals. Furthermore, splenocytes from immunized H1-4RKO mice produce significantly less IFN-γ compared to WT mice. Taken together these data support the existence of a novel HA signaling pathway in regulating EAE susceptibility.
Role of IFI202b in the suppressive ability of CD8+Treg induced in (NZB x NZW) F1 lupus mice (143.56)
Authors: Singh Ram , Dinesh Ravi , Hahn Bevra 
Published: January 2023

Abstract
Tolerance with an artificial peptide (pConsensus, pCons) based on anti-DNA IgG sequences containing MHC class I and class II T cell determinants, causes NZB/NZW F1 female (BWF1) mice to develop CD8+Foxp3+ inhibitory T (Ti) cells which suppress anti-DNA Ig production. CD8+T cells from mice tolerized with pCons expressed Ifi202b more than two-fold higher than cells from untolerized mice, with an increase 1-4 weeks after tolerization and a return toward baseline at 6 weeks. In vitro polyclonal activation significantly increased Ifi202b mRNA expression in tolerized CD8+T cells. Importantly, silencing of Ifi202b abrogated the suppressive capacity of tolerized CD8+T cells. This was associated with decreased expression of Foxp3, and decreased gene and protein expression of TGFb, but not of IL-2, IFN-g, IL-10 or IL-17. These changes were not related with resistance to apoptosis induced in CD8+Treg. Although Ifi202b is induced by interferons, silencing of another IFN-induced gene upregulated in tolerized CD8+T cells, IFNar1, had no effect on the ability of CD8+T cells to suppress autoantibody production. Taken together, these data identify a role for Ifi202b in the suppressive capacity of peptide-induced regulatory CD8+T cells via effects on the expression of Foxp3 and the synthesis of TGFb.
Deimmunization of HLA-DR Epitopes in the C2 Domain of Human FVIII (144.29)
Authors: Song Chang , Martin William , DeGroot Annie , Scott David 
Published: January 2023

Abstract
To reduce immunogenicity of therapeutic proteins, recombinant proteins can be modified to lower HLA binding of epitopes, a method (deimmunization) useful with monoclonal antibodies. Since the immune response to human clotting factor VIIII limits therapeutic efficacy, we reported that immunodominant peptides in the C2 domain of fVIII could be modified to reduce MHCII binding to display less antigenicity in vitro in a hemophilic fVIII KO mouse model (H-2b). We have extended these studies in humanized HLA-DR transgenic mice and identified responses to C2 peptides p2191-2210, p2231-51, p2254-69, p2271-89; and p2310-30 in DR2, DR3, DR4 mice, as well as in DR3 mice crossed to fVIII KO hemophilic mice. DR transgenic mice were immunized with the designated original peptides (ORG) or with rFVIII in CFA, and LN cells tested in T-cell proliferation assays against rFVIII and ORG peptides. While p2191 dominantly stimulates H-2b, other peptides are immunodominant in DR2, DR3 and DR4 mice. A different pattern was revealed (DR3 x fVIII KO) mice depending on whether they also express H-2b. Efforts to test the reactivity/antigencity and immunogenicity of modified (MOD) peptides predicted to have lower class II binding will be reported. These results will be utilized to design and produce a functional full-length modified rFVIII that can be used as a therapeutic protein in hemophilia A (Supported by NIH R43 HL088834-01).
Unique enzymes improve mass spectroscopy of IgG and make it possible to use affinity purification of acid sensitive IgG molecules (144.31)
Authors: Frediksson Sarah , Hedström Martin , Olsson Fredrik , Allhorn Maria , Collin Mattias 
Published: January 2023

Abstract
The human pathogen Streptococcus pyogenes produces various surface proteins in order to avoid the host immune defence systems. Two of these proteins, EndoS and IdeS, have proven to be very useful as tools in antibody engineering. EndoS hydrolyses the chitobiose core of the aspargine linked glycan on the heavy chain of IgG and IdeS is a cysteine protease that cleaves the IgG molecule at a unique site in the hinge region. Here we show, for the first time, that mass spectrometric analysis of antibody fragments are greatly facilitated by combining EndoS and IdeS activities. Utilization of RPC18 LC-ESI-MS generated a well-defined protein envelope with a deconvoluted mass of 24137 Da corresponding to the IgG1 heavy chain cleaved at position Gly236. This opens up new possibilities for utilizing a combination of these enzymes for fast and accurate analysis of IgG:s in a variety of mass spectrometry applications. In addition, a mutated variant of the EndoS enzyme was found to show a high affinity for IgG molecules and has been evaluated in affinity purification of intact, native IgG molecules since it excludes non-native IgG molecules. Furthermore, the affinity purification using the mutant EndoS can be performed at neutral pH which is favourable in purification of acid sensitive IgG clones. The performance and characteristics of Ides, EndoS and the EndoS mutant is highlighted during the presentation with data from several individual studies.
Rare Event detection using Acoustic Cytometry (144.4)
Authors: Anderson April , Bradford Jolene 
Published: January 2023

Abstract
Detection of rare events includes populations comprising less than 5% of the total cells, which involves the detection of stem cells, minimal residual disease and fetomaternal hemorrhage. Analysis of rare cell populations requires the collection of high numbers of events in order to attain a reliable measure of accuracy, leading to long acquisition times. Acoustic cytometry is a new technology which allows dilute samples to be processed quickly. We compared collection times of the Applied Biosystems® Attune™ Acoustic Focusing Cytometer to conventional cytometers. Varying numbers of CD34+ KG-1a cells were spiked into whole blood collected from a normal donor, stained with CD45 & CD34 conjugates, and then collected on traditional and acoustic cytometers. Two hundred thousand live cells were acquired and acquisition time recorded, with results compared across all instruments tested. Rare CD34+ populations were detected on all instruments, giving equivalent results. Acquisition time was significantly reduced using an acoustic cytometer compared to a standard flow cytometer. Acquisition times on the acoustic cytometer can be reduced by up to 5-10 fold compared to traditional flow cytometers, while maintaining comparable results in rare event detection. Utilizing the acoustic focusing cytometer rare event acquisition can be accomplished in one fifth of the time without sacrificing data integrity.
DN-Treg cells induce long-term cardiac allograft survival in fully MHC mismatched recipients but do not eliminate T memory cells (145.13)
Authors: Zhang Zhu-Xu , Lian Dameng , Huang Xuyan , Wang Shuang , Sun Hongtao , Liu Weihua , Min Wei-ping , Garcia Bertha , Jevnikar Anthony 
Published: January 2023

Abstract
Establishment of immune tolerance is a major goal in transplantation. Regulatory T (Treg) cells play an important role in the regulation of immune responses. Our previous studies have shown that TCRαβ+CD3+CD4-CD8-NK1.1- (double negative, DN) Treg cells suppress anti-donor T cell responses and prolong allograft survival in a single MHC-mismatched mouse model. In this study, we tested whether DN-Treg cells can induce long-term graft survival in the stringent BALB/c to C57BL/6 heart transplant model. Adoptive transfer of 107 DN-Treg cells in combination with rapamycin (RAPA) treatment (day 1- 9) induced long-term graft survival (101 vs. 39 days RAPA alone, p&lt;0.01). DN-Treg treated recipients had few graft-infiltrating CD4+ and CD8+ T cells on day 40 compared to massive infiltration in RAPA treated controls. Memory T (Tm) cells (CD3+CD44+) were found to be resistant to DN-Treg cell-mediated suppression in vitro. The effect of DN-Treg on Tm in vivo was tested in B6-Rag1-/- mice. DN-Treg cells along with CD44+ Tm cells modestly prolonged BALB/c skin graft survival in contrast to DN-Treg with CD44- T cells (26 vs 43 days, P&lt;0.001). In conclusion, DN-Treg cells combined with short term immunosuppression, can prolong allograft survival in the absence of tolerance. Furthermore, this may be related to the resistance of Tm cells to DN-Treg cell suppression, suggesting alternative strategies will be needed to control Tm cells and establish tolerance after transplantation.
Establishment of a novel in vitro gut epithelial culture system for the study of T cell mediated gut injury during early GVHD (145.15)
Authors: Matos Jennifer , Dziopa Eugenia , Friedman Thea , Korngold Robert 
Published: January 2023

Abstract
Graft-versus-host disease (GVHD) is the primary complication of blood and marrow transplantion used to treat hematological diseases. Epithelial cells are apoptotic targets during GVHD. Gut epithelium is a key factor in the progression from localized to systemic GVHD, as damage to the intestines allows endotoxin transfer into general circulation, triggering cytokine release, organ damage and systemic inflammation. However, cellular mechanisms that regulate tissue damage in GVHD are poorly understood. Using the established MHC-matched, miHA-mismatched B6→BALB.B irradiation model of GVHD, we generated a novel primary cell culture system for in vitro study of gut epithelial cell injury by extracting intestinal epithelial cells from BALB.B mice for co-culture with B6 T cells. Our cultures are representative in their heterogeneity, containing both cytokeratin 15+ epithelial cells and smooth muscle cells that are easily differentiated from one another for analysis, with no contaminating dendritic cells or fibroblasts. In the skin, CD4+ and CD8+ T cells target cytokeratin 15+ stem cells of the basal membrane. Therefore, our in vitro gut epithelial cells may be targeted, and apoptosis induced, as in vivo. With this apoptotic co-culture model system in place, we will determine which of the perforin/granzyme, TRAIL, or Fas/FasL apoptotic pathways is critical at the early stages of intestinal GVHD, providing insight into potential therapeutic targets for prevention or treatment of GVHD.