bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcriptional regulation of the human FCGR2B gene mediated by promoter polymorphisms associated with Systemic Lupus Erythematosus (35.38)
Authors: Olferiev Mikhail , Masuda Emi , Blank Marissa C , Pricop Luminita 
Published: January 2023

Abstract
The inihibitory Fc gamma receptor IIb is an important negative regulator of peripheral tolerance. The [−343 G to C] SNP in the human FCGR2B promoter is associated with Systemic lupus erythematosus (SLE). The mutant promoter [−343C/C] hasdecreased transcriptional activity. Analyses by electromobility shift, chromatin immunoprecipitation and promoter pull-down assays demonstrated binding of AP1 factors to the FCGR2B promoter. The binding of AP1 factors was decreased with the mutant FCGR2B promoter compared with the common promoter. The −343 G to C substitution enabled the specific interaction of the transcription factor Yin-Yang 1 with the mutant FCGR2B promoter. Yin-Yang 1 competed with AP1 for binding at the −343 site, and contributed to the repression of the mutant FCGR2B promoter activity. The [−343 C/C] genotype was found to be in linkage disequilibrium with the [−77 T to A] SNP relative to the transcription initiation site. The [−77 A/A] promoter constructs displayed increased activity compared with [−77 T/T] promoter constructs in luciferase assays, suggesting a role for the −77 SNP in the transactivation of the FCGR2B promoter. Allelic variations at positions −343 and −77 identified in the human FCGR2B promoter differentially modulate the transcriptional activity and may play a role in the expression and progression of SLE disease.
Norepinephrine mediated PKA-dependent regulation of Forkhead transcription factors in murine S49 thymoma cells (35.42)
Authors: Davis Daniel Hal , Graf Lloyd H , Cohen Rhonna L , Chambers Donald A 
Published: January 2023

Abstract
Previous investigations have revealed the presence of a norepinephrine/β2-adrenergic receptor/cAMP/PKA-dependent mRNA decay system in T lymphocytes. The Fox family of transcription factors has been implicated in regulating lymphocyte physiology. Foxm1b is reported to be a master gene related to cellular proliferation and Foxo1 has been associated with the suppression of proliferation and promotion of apoptosis. Therefore, we explored whether a PKA-selective cAMP analogue (8Br-cAMP) and NE could regulate BALB/c murine thymocyte cell lines, S49wt and S49kin- (PKA deficient), by affecting the genetic expression of Foxm1b and Foxo1. Real-time PCR studies revealed a 2–3 fold upregulation of Foxo1 mRNA in S49wt, but not kin- cells with 150uM NE or 600uM cAMP as early as 2 hrs after treatment. Foxm1b mRNA was downregulated in S49wt cells by greater than 2 fold by 8Br-cAMP after 6 hrs (at 24 hrs). Treatment with an Epac-selective cAMP (500uM) analogue resulted in no change in Foxo1 or Foxm1b expression in S49 wt or S49 kin- cells. These results associate PKA in modulating both increased Foxo1 mRNA expression and decreased Foxm1b expression and consequent fox gene-mediated regulation of lymphocyte physiology.
Supported by NIH.
Identification a novel NF-kB regulator by tandem affinity purification combined with mass spectrometry (35.44)
Authors: Wan Fengyi , Zheng Lixin , Anderson D. Eric , Lam Lloyd T. , Hegde Vijay , Deutsch Walter A. , Staudt Louis M. , Lenardo Michael J. 
Published: January 2023

Abstract
The molecular events involved in NF-kB signal transduction from the cell surface to the nucleus have been extensively studied over the last two decades. However, the mechanism of recruitment of NF-kB to target genes and NF-kB-induced transcriptional events after recruitment remains enigmatic. The p65-p50 heterodimer is the most abundant form of active NF-kB. The crystal structure of the p65-p50 heterodimer bound to DNA revealed that p65 binds to the 3′ half of kB sites where most of the kB sequence variation occurs, while p50 binds to the relatively invariant 5′ half. In an effort to identify novel regulatory factors of NF-B, we used p65 as bait protein and performed tandem affinity purification combined with mass spectrometry on mammalian cell extracts. Through this approach, we identified a novel regulatory protein that specifically interacts with p65. RNA interference experiments revealed that knockdown of such protein specifically inhibited p65-mediated transcription of various target genes. This finding may shed light on the mechanism of p65 regulation and provide a new insight into NF-kB specificity.
Physical interaction between the IgH 3′ regulatory region and its target, the expressed VH gene (35.47)
Authors: Ju Zhongliang , Birshtein Barbara K. 
Published: January 2023

Abstract
Immunoglobulin heavy chain (IgH) gene expression is regulated by the intronic enhancer (Eμ) and a complex regulatory region located 3′ of the IgH gene locus (3′RR). One mechanism by which the 3′ RR may act is by physical interaction between 3′RR enhancers and the targeted VH sequence. Using the chromosome conformation capture (3C) assay, we detected interactions between the 3′RR and the expressed VH gene in MPC-11 plasmacytoma cells. The interactions did not occur in MEL cells in which the IgH locus is silent and did not extend to the hole gene, which is the nearest downstream non-Ig gene, nor to the calreticulin gene located on another chromosome. Eμ is not required for this interaction because we observed loops in 9921 cells, an MPC-11 variant in which Eμ has been deleted. Furthermore, we found that 3′RR regulators interacted with each other. The translocated c-myc gene in MPC-11 interacted with the 3′RR, but not with Eμ located on the other, i.e. IgH expressed, allele. This implies that the VH-3′RR interaction occurred in cis but not trans. We observed a similar VH-3′RR interaction spanning ~200kb in resting splenic B cells. We conclude that the 3′RR physically interacts with its target VH gene in both splenic B cells and plasma cells. We predict that this interaction provides a scaffold on which the 3′RR can act on otherwise linearly distant target sequences to influence IgH expression.
Supported by NIH AI13509.
A Large-scale survey of human Ig Heavy Chain genes in Genbank: Structural Correlates of Autoreactivity and Statistical Signatures of Molecular Mechanisms (35.5)
Authors: Volpe Joseph M , Kepler Thomas B 
Published: January 2023

Abstract
There are over 20,000 DNA sequences from human immunoglobulins represented in Genbank. This wealth of DNA sequence information provides an opportunity to study the structural correlates of immunoglobulin function, including autoreactivity and to infer molecular mechanism from statistical signatures. After careful control for removal of clonally related genes, we analyzed approximately 9,400 productively rearranged sequences in order to establish baseline statistical characteristics, such as gene segment usage, n-nucleotide and mutation frequency, and the biochemical properties of CDR regions. We then analyzed 181 autoreactive and 64 anti-DNA Ig sequences against this baseline to discern statistical differences that might distinguish structurally these functionally distinct genes. Our data reveal that there are indeed distinctive biases in gene segment usage, CDR3 length, and mutation frequency that differ from one group to another. We also fit data on n-nucleotide frequency to a negative binomial model, which reveals striking differences between the DJ and VD junctions. These results have an appealing interpretation in terms of differential access of terminal deoxynucleotidyl transferase in the developing junctions.
Characterization of MHC-encoded genes in the chicken governing NK cell responses (35.52)
Authors: Zhang Lei , Goto Ronald M , Goebel Thomas , Yokoyama Wayne M , Miller Marcia M 
Published: January 2023

Abstract
Little is known about the molecular interactions that control NK cell responses in avian species. To define NK cells and their interactions in the chicken we are starting with a gene (Blec2) found within the MHC-B region that encodes a putative inhibitory c-type NK cell receptor. We prepared reporter cells expressing a transfected chimeric receptor molecule having the ectodomain derived from Blec2 (BAZ cells) to interrogate a panel of cell lines. We found that BAZ cells were reliably stimulated by only two cell lines. LMH cells induce a robust BAZ response that is inhibited in a dose response manner by pretreatment with tunicamycin. BAZ cells are also stimulated by RP9 cells expressing high levels of a transfected non-classical MHCI allele (YF 7.1) originating from MHC-Y. In contrast RP9 cells expressing two different classical MHCI alleles (BF2*13 and BF2*21) failed to stimulate the BAZ reporters. In cytotoxicity assays the YF7.1 allele also inhibits RP9 cell killing by IL-2-stimulated CT8+ NK cells isolated from embryonic spleens. Hence non-classical class I molecules encoded in MHC-Y may be functionally active ligands for NK cell inhibitory receptors. N-linked carbohydrate may contribute to the recognition process. These results support the idea that MHC-Y and MHC-B are linked in cooperative interactions governing NK cell responses.
Evidence for Ku70/Ku80 association with full length RAG1 (35.6)
Authors: Raval Prafulla , Kumar Sushil , Kriatchko Aleksei N. , Swanson Patrick C. 
Published: January 2023

Abstract
Antigen receptor genes are assembled by a form of site-specific DNA rearrangement termed V(D)J recombination. This process proceeds through two distinct phases: a cleavage phase in which the RAG1 and RAG2 proteins introduce DNA double-strand breaks at recombination signal sequences (RSSs), and a joining phase in which the resulting DNA breaks are processed and repaired via the non-homologous end-joining (NHEJ) repair pathway. Genetic and biochemical evidence suggest that the RAG proteins play an active role in guiding the repair of DNA breaks introduced during V(D)J recombination to the NHEJ pathway. However, evidence for specific association between the RAG proteins and any of the factors involved in NHEJ remains elusive. Here we present biochemical evidence that two components of the NHEJ pathway, Ku70 and Ku80, interact with full-length RAG1 and can become integrated into a stable RAG-RSS complex assembled with full-length RAG1 and core RAG2, but not core RAG1 (aa 384–1040) and either core or full-length RAG2. Formation of this complex minimally requires residues 211–1040 of RAG1. These results provide a biochemical link between the two phases of V(D)J recombination.
This research is supported by National Institutes of Health grant 1R01 AI055599 to P.C.S.
RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination (35.8)
Authors: Matthews Adam Goon Wai , Kuo Alex , Ramon-Maiques Santiago , Han Sunmi , Gallardo Mercedes , Yang Wei , Gozani Or , Oettinger Marjorie 
Published: January 2023

Abstract
A wide range of studies suggest that V(D)J recombination is regulated by modulation of the chromatin structure of antigen receptor loci during lymphoid development. Recent work has analyzed the pattern of histone modifications present at the immunoglobulin heavy chain (IgH) locus during various stages of B cell development. However, mechanisms linking alterations in histone modifications to the function of the RAG recombinase have remained elusive. Here, we show that the RAG2 plant homeodomain (PHD) finger specifically interacts with histone H3 trimethylated at lysine 4 (H3K4me3). The RAG2 PHD finger is necessary and sufficient for H3K4me3 peptide binding. A high-resolution crystal structure of the RAG2 PHD finger bound to H3K4me3 reveals that the trimethyl-K4 moiety is bound in a groove of three hydrophobic residues. Mutations that abrogate recognition of H3K4me3 severely impair V(D)J recombination in vivo without affecting V(D)J cleavage in vitro. Notably, a conserved tryptophan residue present within the RAG2 PHD finger and mutated in patients with immunodeficiency syndromes is critical for RAG2’s recognition of methylated H3. Together our results identify a novel function for histone methylation in DNA recombination. In addition, we provide the first evidence that disrupting the read-out of histone modifications can cause an inherited human disease.
This work was supported by NIH grant R01-GM48026. A.G.W.M. is a Howard Hughes Medical Institute Predoctoral Fellow.
Liver plasmacytoid dendritic cells stimulate antigen specific T cells in vivo (36.16)
Authors: Kingham T. Peter , Plitas George , Chaudhry Umer I. , Stableford Jennifer , DeMatteo Ronald P. 
Published: January 2023

Abstract
Introduction: Liver plasmacytoid dendritic cells (pDC) are considered to be weak stimulators of in vitro T cell responses. We sought to determine their ability to prime an antigen specific T cell response in vivo.
Methods: C57BL/6 murine spleen and liver pDC (CD11c+B220+NK1.1−CD19−CD11b−) were expanded in vivo with the use of an adenovirus encoding murine Flt3L cDNA and isolated by fluorescence activated cell sorting. CD45.2+ CFSE-labeled OT-I T cells were injected in the lateral tail vein of congenic recipient CD45.1 mice, pDC loaded with OVA257–264 (1μg/ml) or TRP-2181–188 (1μg/ml) were subsequently injected into the footpad. Three days later popliteal nodal lymphocytes were analyzed by flow cytometry for dissolution of CFSE fluorescence among CD3+CD8+CD45.2+cells.
Results: Flt3L expanded OVA257–264 loaded liver pDC and spleen pDC caused 70% and 79% respectively of the transferred T cells to divide. TRP-2181–188 loaded pDC induced no T cell division.
Conclusion: Liver pDC can initiate antigen specific T cell responses in vivo.
This work was supported by grant DK068346.
Functional dichotomy of plasmacytoid dendritic cells: Production of type I interferon versus specific activation of T cells (36.17)
Authors: Jähn Peter Siegfried , Schmitz Jürgen , Dzionek Andrzej 
Published: January 2023

Abstract
Here we describe a remarkable functional dichotomy of plasmacytoid dendritic cells (PDC): dependent on stimulation, PDC produce either type I interferon (IFN-I) and express the programmed cell death receptor 1 ligand 1 (CD274) or efficiently present antigen (Ag) to T cells. Upon culturing, PDC spontaneously redistribute MHC class II molecules from non-lysosomal to lysosomal compartments, resembling the so-called MHC-II-enriched compartments (MIIC). Cytomegalovirus (CMV)-Ag that is targeted to PDC using a CD303 (BDCA-2) mAb is rapidly endocytosed and traffics via early sorting endosomes to emerging MIIC. Type B CpG oligonucleotide (CpG-B)-stimulated PDC efficiently present CMV-Ag and are thus capable of stimulating CMV-specific T helper cells. CpG-A-stimulated PDC produce IFN-I and express CD274. Induction of Ag presentation by CpG-B inhibits induction of IFN-I production by CpG-A. IFN-I itself induces CD274 expression in PDC, indicating that induction of CD274 expression by CpG-A is indirectly mediated by induction of IFN-I production. Our results suggest that Ag presentation and production of IFN-I are mutually exclusively induced. This may be critical for controlling adaptive immunity, maintaining self-tolerance and preventing autoimmunity.
Chronic ethanol ingestion induces peripheral tolerance by dendritic cells (36.21)
Authors: Fan Ji , Schlueter Annette J 
Published: January 2023

Abstract
As initiators of immune responses, dendritic cells (DCs) are required for antigen specific activation of na·ve T cells in the defense against infectious agents. The immune dysfunction in chronic alcoholics could be due to impaired immunostimulatory function or enhanced regulatory function of DCs by exposure to ethanol. Using a murine model of alcoholism, we previously assessed the ability of ethanol exposed DCs to stimulate T cell proliferation. (In this model, C57Bl/6 mice were administrated 20% ethanol in their drinking water, and maintained for 4 or 16 weeks without evidence of stress reactions.) DC chronically exposed to EtOH for 4 or 16 weeks were found to have decreased DC antigen presenting ability and induced hyporesponsiveness of allogenic T cells in mixed leukocyte reaction. In order to understand what mechanism was behind this phenomenon, we investigated whether regulatory T cells could be induced by DC from the same murine model of alcoholism. CFSE labeled splenocytes from OTII mice were transferred to syngeneic recipient mice on day 1. On day2 fresh splenic DC were purified on autoMACS, loaded with OVA peptide 329–337, and transferred to the same recipient mice. On day 4, the CD4+Foxp3+ T cell compartment in CFSE labeled (OTII) splenocytes was defined using flow cytometry. The results showed regulatory T cells were increased in response to contact with EtOH exposed DC. Meanwhile IL10 production by OTII CD4 T cells increased and IFN-gamma production decreased. We also found in EtOH exposed DC that PIR A/B expression (a regulatory DC marker) was increased, which might explain why EtOH exposed DC could induce peripheral tolerance as shown above. Additional phenotypic and functional characterization of these EtOH induced rDC subsets are in progress.
Supported by NIH RO1 AA014405 and AA014406.
P2X7 Activation Induces Novel Dendritic Cell Filopodia (36.23)
Authors: Thomas Louis Michael , Chu Jessica , Watkins Simon C. , Salter Russell D. 
Published: January 2023

Abstract
Dendritic cells (DC) express multiple surface purinergic receptors that bind extracellular adenosine triphosphate (ATPe) resulting in complex changes in cell morphology and function. We demonstrate that ATPe induces rapid extension of filopodia from cultured DC that are distinct from pre-existing dendritic processes. These processes were not observed in other cell types analyzed and require the activity of P2X7 purinergic receptors, based on pharmacologic studies using receptor agonists and antagonists, and on loss of filopodia formation in a mutant DC cell line selected to be deficient in P2X7 expression. Phosphatidylinositol 4-kinase III α, which associates with P2X7, participates in filopodia formation based on inhibition by wortmannin at high concentrations that inhibit enzyme activity. Over time, filopodia often attain a beaded appearance with subsequent detachment of microvesicles from the cell. We hypothesize that P2X7 stimulation by ATPe could facilitate release of cell-associated antigens within microvesicles that might be internalized by phagocytes including DC, and therefore represent a novel cross-presentation pathway.
A method for rapid isolation of highly purified human monocytes using fully automated negative cell selection (36.25)
Authors: Yuan Ning , Guilbault Benoit G , Milton Graeme T , Fadum Jodie , Damen Jackie E , Eaves Allen C , Thomas Terry E , Wognum Albertus W 
Published: January 2023

Abstract
The preparation of highly purified monocytes for experimentation has traditionally been difficult, requiring multiple steps and many hours of work. We report the successful development of a rapid negative selection method that enables the preparation of highly purified monocytes from human peripheral blood using EasySep® column-free immunomagnetic cell separation technology. A cocktail of monoclonal antibodies incorporated into tetrameric antibody complexes was used to crosslink unwanted cells in the sample to EasySep® magnetic particles. The tube containing the labelled cell suspension was then placed in an EasySep® magnet for 2.5 minutes. Unlabelled cells were recovered by pouring off the cell suspension while labelled unwanted cells were held to the walls of the tube by the magnetic field. The whole procedure was completed in 30 minutes and yielded CD14+CD16− monocyte fractions that were on average 90% pure with an average recovery above 60%. Stimulation of purified monocytes with GM-CSF, IL-4 and LPS led to efficient differentiation into dendritic cells (DC) as identified by the expression of the DC markers CD1a and CD83, and the loss of the monocyte marker CD14. Differentiated DC induced robust allogeneic CD4+ T cell proliferation, confirming that the isolated monocytes were fully competent to differentiate into functional DCs. This method was also fully automated using the RoboSep® cell separator.
Tracking antigenic intermediates throughout the antigen processing pathway during Listeria monocytogenes infection (36.3)
Authors: Shugart Jessica Alis , Shastri Nilabh 
Published: January 2023

Abstract
Prior to their appearance on the cell surface where they are presented by MHC I molecules to T cells, antigenic peptides are created by a complex array of intracellular processing events. Using conventional T cell assays, only the final peptide can be efficiently detected on the cell surface. In order to detect the full range of antigenic intermediates that exist intracellularly throughout the antigen processing pathway, members of the Shastri lab have successfully developed the KOVAK system. KOVAK is a modified form of ovalbumin which contains enzymatically cleavable residues flanking the antigenic peptide, SHL8, thus allowing it to be liberated from the context of any antigenic intermediate and detected by SHL8-specific T cells following HPLC fractionation of cell extracts. Over the past year, we have utilized this system to track antigenic intermediates in the processing of KOVAK expressed by Listeria monocytogenes. Because Listeria enters the cell phagosomally and then injects itself into the cytoplasm, Listeria infection provides an excellent model to investigate the roles of various components in both the exogenous and endogenous pathways, and where these pathways merge intracellularly. So far we have successfully used this model in combination with knockout mice and inhibitors to delineate the roles of phagosomal proteases, cytoplasmic proteases and the proteasome, chaperones, TAP transport, and ERAAP in the processing of Listeria-derived antigens in infected macrophages. We will also investigate the roles of macrophage and dendritic cell maturation in the processing of Listeria-derived antigens.
In vitro vaccination site: a novel test bed for immunopotentiators (36.30)
Authors: Ma Yifan , Pawar Santosh , Sanchez-Schmitz Guzman , Poisson Louis , Byers Anthony , Schanen Brian C , Warren William L , Mishkin Eric M , Drake Don R , Higbee Russell G 
Published: January 2023

Abstract
Cytokine-derived dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) are widely used for adjuvant validation. However, the functional limitations reduce their predictive value. A 3D endothelial cell/collagen culture called “vaccination site (VS)” has been characterized as a novel in vitro system, which resembles in vivo physiological conditions and autonomously generates DCs. We used the VS model to investigate the immunopotency of TLR agonists (Poly IC, Gardiquimod, CpG 2006, and LPS). The administration of TLR agonists in the VS for 24 hr induced a wide range of cytokines, including IL-1 alpha/beta, IL-6, IL-8, IL-10, and TNF. The adjuvant-elicited cytokine profiles suggested a relative immunopotency: LPS&gt; Gardiquimod&gt; Poly IC≥ CpG 2006. Compared with the PBMC culture, the VS produced 2–100 fold more cytokines, indicating its enhanced sensitivity in response to TLR agonists. In addition to inducing cytokine production, TLR agonists (e.g. Gardiquimod) increased the generation of VS-derived DCs and elevated CD86 expression. Moreover, VS-derived APCs treated with TLR agonists facilitated T-cell proliferation and augmented Tetanus toxoid-specific antibody production. These data indicate that TLR agonists improved the antigen-presenting capability of VS-derived APCs. Overall the VS model is a more efficient test bed than the traditional PBMC culture to evaluate adjuvant candidates.
(Work supported by DARPA Rapid Vaccine Assessment Program)