bluuebunny/crossref_metadata_2025_split huggingface dataset:
Extensive Junctional Diversity of Ig Heavy Chain Rearrangements Generated in the Progeny of Single Fetal Multipotent Hematopoietic Cells in the Absence of Selection
Authors: Nourrit Françoise , Doyen Noëlle , Kourilsky Philippe , Rougeon François , Cumano Ana 
Published: January 2023

AbstractWe analyzed the progeny of individual multipotent hemopoietic cells, derived from the para-aortic splanchopleura, the earliest identified source of lymphocyte precursors in pre-liver mouse embryos. Single precursors were expanded in an in vitro culture system that permits both commitment and differentiation of B cell precursors. We show that from one single multipotent progenitor we could obtain large numbers of B cell precursors that rearrange the Ig heavy chain genes and generate a repertoire as diverse as that observed in adult populations. N region additions are present at V(D)J junctions, showing that terminal deoxynucleotidyl transferase expression has been switched on and is not, consequently, an intrinsic property of adult stem cells. Throughout the culture period, cells show a majority of DJ vs V(D)J rearrangements and a ratio of 2:1 of nonproductive to productive V(D)J rearrangements, which is close to the expected frequency in the absence of selection. In addition, counterselection for D-J rearrangements in reading frame 2 is observed in V(D)J joints, and allelic exclusion was consistently observed. We conclude that of the three events associated with heavy chain rearrangement, two of them, namely allelic exclusion and counterselection of cells in which the D segment is in reading frame 2, are intrinsic to the cell, while selection of productive heavy chain rearrangements is induced in the bone marrow environment.
Novel Glycosylation of HLA-DRα Disrupts Antigen Presentation Without Altering Endosomal Localization
Authors: Guerra Carolyn B. , Busch Robert , Doebele Robert C. , Liu Wendy , Sawada Tetsuji , Kwok William W. , Chang Ming-der Y. , Mellins Elizabeth D. 
Published: January 2023

AbstractThe HLA-DR hemizygous B lymphoblastoid cell line, 10.24.6, has a DRA mutation (Pro96→Ser) that creates a novel glycosylation site at Asn94. The mutant DR molecules are primarily associated with nested fragments of invariant chain (class II-associated invariant chain peptides), and their interaction with HLA-DM is impaired. Here we further analyzed the defect in 10.24.6 cells. Expressing Ser96 mutant DRA cDNA in DRA-null cells recapitulated the 10.24.6 phenotype, indicating that the mutation causes the Ag presentation defect. A mutation to Ala96α, which does not introduce an extra glycan, generated a normal phenotype; the critical role of the glycan was further supported by experiments in which N-glycosylation was blocked by tunicamycin. We also evaluated whether the 10.24.6 mutation affected DR3 maturation or trafficking. Metabolic labeling and subcellular fractionation showed that assembly, endosomal transport, and invariant chain proteolysis of mutant DR3 molecules were similar to wild-type. A slight delay in export from the endoplasmic reticulum to the Golgi apparatus in 10.24.6 cells probably did not contribute significantly to the Ag presentation defect, because the abundance of DM and mutant DR in peptide-loading compartments was normal at steady state. Our results indicate that proper localization of these molecules does not depend on their interaction.
IL-15 Enhances the Response of Human γδ T Cells to Nonpetide Microbial Antigens
Authors: García Verónica E. , Jullien Denis , Song Mark , Uyemura Koichi , Shuai Ke , Morita Craig T. , Modlin Robert L. 
Published: January 2023

AbstractHuman γδ T cells have the ability to rapidly expand and produce IFN-γ in response to nonpeptide Ags of microbial pathogens, in particular a class of compounds known as the prenyl phosphates. We investigated the ability of IL-15, a T cell growth factor, to modulate prenyl phosphate-induced γδ T cell proliferation and cytokine production. IL-15 significantly enhanced the expansion of γδ T cells in the peripheral blood after stimulation in vitro with isopentenyl pyrophosphate. Moreover, using γδ T cell clones, we determined that IL-15-induced T cell proliferation was dependent on the IL-2Rβ chain but not the IL-2Rα chain. We therefore studied the IL-15Rα chain expression in human γδ T cells in the presence or absence of nonpeptide Ags. We found IL-15Rα mRNA expression in IL-15-stimulated and Ag-stimulated human γδ T cells but not in resting γδ T cells. Although IL-15 itself had little effect on the production of IFN-γ, IL-15 plus IL-12 acted synergistically to augment IFN-γ production by γδ T cells. Moreover, we showed that this increase in IFN-γ could be explained by the dual activation of STAT1 and STAT4 by IL-15 and IL-12, respectively. Taken together, these results suggest that IL-15 may contribute to activation of human γδ T cells in the immune response to microbial pathogens.
Dominant Signals Leading to Inhibitor κB Protein Degradation Mediate CD40 Ligand Rescue of WEHI 231 Immature B Cells from Receptor-Mediated Apoptosis
Authors: Schauer Stephanie L. , Bellas Robert E. , Sonenshein Gail E. 
Published: January 2023

Abstract
Recently, we demonstrated maintenance of nuclear factor (NF)-κB/Rel factors plays a major role in B cell survival. Treatment of WEHI 231 immature B cells with an Ab against the surface IgM protein (anti-IgM) induces apoptosis that can be rescued by engagement of CD40 receptor. The dramatic decrease in high basal levels of NF-κB/Rel activity induced by anti-IgM treatment led to cell death. CD40 ligand (CD40L) treatment prevented the drop in NF-κB/Rel factor binding by inducing a sustained decrease in inhibitor (I) κB-α and transient decrease in IκB-β protein levels. In this study, we have investigated the regulation of these NF-κB/Rel-inhibitory proteins. In exponentially growing WEHI 231 cells, the IκB-α and IκB-β proteins decayed with an approximate t1/2 of 38 and 76 min, respectively, which was blocked effectively upon addition of the proteasome-specific inhibitor (benzylcarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO). Anti-IgM treatment stabilized IκB-α and IκB-β proteins. CD40L treatment resulted in a dramatic decrease in t1/2 (&lt;5 min) for both IκB molecules, which was inhibited by addition of Z-LLF-CHO. CD40L treatment also caused a delayed increase in IκB-β mRNA levels, most likely contributing to the observed recovery of IκB-β levels. Microinjection of IκB-α-glutathione S-transferase fusion protein into nuclei of WEHI 231 cells ablated protection by CD40L from receptor-mediated killing. Furthermore, CD40L rescued apoptosis induced upon microinjection of a vector expressing wild-type IκB-α, but not a 32A/36A mutant form of IκB-α, unable to be phosphorylated and hence degraded. Thus, control of turnover of IκB proteins by CD40L plays a major role in maintenance of NF-κB/Rel and resultant rescue of WEHI 231 cells from apoptosis.
The 5′ Untranslated Region, Signal Peptide, and the Coding Sequence of the Carboxyl Terminus of IL-15 Participate in Its Multifaceted Translational Control
Authors: Bamford Richard N. , DeFilippis Andrew P. , Azimi Nazli , Kurys Gloria , Waldmann Thomas A. 
Published: January 2023

AbstractWe previously reported that the AUG-burdened 5′ untranslated region (UTR) of IL-15 mRNA impedes its translation. Here we demonstrate that the nucleotide or protein sequences of the IL-15 signal peptide and carboxyl terminus also contribute to the poor translation of IL-15 transcripts. In particular, the exchange of the IL-15 signal peptide coding sequence with that of IL-2 increased cellular and secreted levels of IL-15 protein 15- to 20-fold in COS cells, while IL-2 transcripts with the IL-15 signal peptide generated 30- to 50-fold less IL-2 protein than wild-type IL-2. Furthermore, the addition of an artificial epitope tag to the 3′ coding sequence of IL-15 increased its protein production 5- to 10-fold. Combining these two IL-15 message modifications, in addition to removing the 5′ UTR, increased IL-15 synthesis 250-fold compared with a wild-type construct with an intact 5′ UTR. These data suggest that IL-15 mRNA, unlike IL-2 mRNA, may exist in translationally inactive pools. By storing translationally quiescent IL-15 mRNA, cells might respond to intracellular infections or other stimuli by rapidly transforming IL-15 message into one that can be efficiently translated.
Thrombopoietin (TPO) Knockout Phenotype Induced by Cross-Reactive Antibodies Against TPO Following Injection of Mice with Recombinant Adenovirus Encoding Human TPO
Authors: Abina Mohammed-Amine , Tulliez Micheline , Duffour Marie-Thérèse , Debili Najet , Lacout Catherine , Villeval Jean-Luc , Wendling Françoise , Vainchenker William , Haddada Hedi 
Published: January 2023

Abstract
Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.
Expression and Function of the Type 3 Complement Receptor in Tissues of the Developing Mouse
Authors: Hughes Derralynn A. , Gordon Siamon 
Published: January 2023

Abstract
Macrophage (Mφ) expression of the leukocyte integrins has been implicated in their adhesion and migration in the adult. Little is known, however, of the expression or function of these molecules during development. This study defines the spatial and temporal sequences of expression of the type 3 complement receptor (CR3) in the developing mouse; establishes the functional efficacy of this molecule in spreading, adhesion, and phagocytosis; and investigates its role in inflammatory and constitutive migration. Expression of CR3 on monocytes occurred early compared to Mφ-restricted glycoprotein F4/80, but expression on stellate tissue Mφ appeared later than F4/80 and was transient. Expression of CR3 on resident tissue Mφ is more widespread during development, being retained on only very specific Mφ populations in the adult. Neutrophil polymorphs expressed CR3 from day 17 of gestation onward. The anti-CR3 mAb 5C6 was used to investigate the role of CR3 in adhesion, spreading, and phagocytosis by neonatal Mφ. Neonatal macrophages were found to adhere, spread, and phagocytose by CR3-dependent mechanisms, and a CR3-independent system was implicated in the spreading of neonatal Mφ. The role of CR3 in migration during development was then investigated. 5C6 had potent effects on the early stages of the migration of myelomonocytic cells to an inflammatory stimulus in vivo. Despite efficient transplacental transfer of the Ab from pregnant mother to fetus, the process by which monocytes generate populations of resident tissue Mφ was undisrupted, indicating the existence of CR3-independent mechanisms of monocyte migration during development.
Cationic Lipids Enhance Cytokine and Cell Influx Levels in the Lung Following Administration of Plasmid: Cationic Lipid Complexes
Authors: Freimark Bruce D. , Blezinger H. Paul , Florack Valarie J. , Nordstrom Jeffrey L. , Long Scott D. , Deshpande Deepa S. , Nochumson Sam , Petrak Karel L. 
Published: January 2023

Abstract
Administration of plasmid/lipid complexes to the lung airways may be associated, in addition to expression of transgene, with a range of other responses. We report here the induction of cytokines and cellular influx in the lung airway following intratracheal administration of an N-[1-(2–3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol/plasmid positively charged complex in mice. We show that 1) the appearance of the Th1-associated cytokines IFN-γ and IL-12 in bronchoalveolar lavage fluid is caused by unmethylated CpG dinucleotide sequences present within the plasmid, and is enhanced by the lipid formulation; 2) cationic lipids by themselves do not induce IL-12 or IL-12p40; 3) TNF-α is rapidly induced by cationic lipids and plasmid/lipid complex, but not by plasmid alone; 4) an acute cellular influx is induced by cationic lipid alone and by a plasmid/lipid complex, but to a much lesser extent by plasmid alone; and 5) plasmid methylation does not influence the degree of inflammatory cell influx. The induction of the innate immune responses by plasmid/lipid complexes may be advantageous to gene therapy of lung diseases. In particular, induction of the Th1 cell-promoting cytokines by plasmid/lipid complexes could, in conjunction with an expressed transgene, be used to modulate immune responses in the lung airways in disease conditions that are deficient in Th1 cell responses or that have a dominant Th2 phenotype. Alternatively, the elimination of immunostimulatory sequences in plasmids may improve the tolerability and/or efficacy of nonviral gene therapy, especially for diseases requiring chronic administration.
Dendritic Cells as the Terminal Stage of Monocyte Differentiation
Authors: Palucka Karolina A. , Taquet Nicolas , Sanchez-Chapuis Francoise , Gluckman Jean Claude 
Published: January 2023

Abstract
Monocytes (MO) cultured for ≥5 days with either macrophage-CSF (M-CSF) or granulocyte macrophage (GM)-CSF and IL-4 differentiated without concomitant proliferation into CD14+ macrophages (Mφ) or CD1a+ dendritic cells (DC), respectively. When adherent and nonadherent CD14high Mφ from M-CSF cultures were separated and cultured further in cytokine-free medium or with GM-CSF/IL-4, most cells from both fractions that were exposed to GM-CSF/IL-4 acquired CD1a expression and DC morphology and function. Conversely, GM-CSF/IL-4 withdrawal at day 5 and additional culture of sorted CD1a+ DC for 2 to 7 days in cytokine-free medium led to cells rapidly becoming adherent CD1a−CD14+ Mφ. Replacing GM-CSF/IL-4 with M-CSF hastened the conversion of DC to Mφ without increasing cell numbers. CD1a+CD14−CD83+ mature DC were induced by a ≥2-day exposure to MO-conditioned medium, LPS, or TNF-α/IL-1β. Upon cytokine removal or culture with M-CSF, DC that had been pushed to maturation by conditioned medium or LPS remained stable or died in the new environment. TNF-α/IL-1β-driven DC displayed heterogeneous CD83 expression and could thus be sorted into CD83high and CD83low/− cells; in cytokine-free medium or in M-CSF, most CD83low/− cells converted to Mφ, whereas most CD83high cells remained nonadherent CD1a+CD14− or died and thus appeared truly terminally differentiated. Hence, MO are precursors of Mφ as well as of DC, with each cell type having the capability to convert into the other until late in the differentiation/maturation process. Accordingly, the cytokine environment and the presence of differentiation and/or other stimulatory signals may be the “final decision-making factors” determining whether these cells will acquire DC or Mφ characteristics and function.
Monocyte Chemoattractant Protein-1 Synthesis by Murine Lung Fibroblasts Modulates CD4+ T Cell Activation
Authors: Hogaboam Cory M. , Lukacs Nicholas W. , Chensue Stephen W. , Strieter Robert M. , Kunkel Steven L. 
Published: January 2023

AbstractThis study addressed the role of endogenous monocyte chemoattractant protein-1 (MCP-1) in Ag-stimulated lymphokine synthesis and proliferation by CD4+ T cells during their coculture with purified lung fibroblasts or splenic macrophages. Initial experiments showed that fibroblasts exposed to IL-4, TNF α, or IL-4 and TNF-α (all at 10 ng/ml) for 24 h released five- to eightfold more MCP-1 than similarly treated splenic macrophages. In 72-h coculture experiments, the synthesis of IL-4 by OVA-activated CD4+ T cells added to lung fibroblasts or splenic macrophages was significantly inhibited when endogenous MCP-1 was neutralized using polyclonal anti-MCP-1 antiserum. In these same cocultures, IFN-γ levels were significantly enhanced. Similarly, IFN-γ levels were significantly enhanced in 72-h cocultures of a purified peptide derivative-activated CD4+ Th1 clone and lung fibroblasts or splenic macrophages following immunoneutralization of MCP-1. In separate experiments, the selective inhibition of MCP-1 synthesis by lung fibroblasts and splenic macrophages using an MCP-1 antisense oligonucleotide significantly enhanced the proliferation of CD4+ T cells during a 96-h coculture. Taken together, these data suggest that MCP-1 exerts an immunomodulatory effect on CD4+ T cell-derived IL-4 and IFN-γ release and CD4+ T cell proliferation during cell-to-cell interactions.
In Vitro Stimulation of C1s Proteolytic Activities by C1s-Presenting Autoantibodies from Patients with Systemic Lupus Erythematosus
Authors: He Shiping , Lin Ya-Ling 
Published: January 2023

Abstract
Anti-C1s autoantibodies (IgG forms), which recognize the conjunction of C1s heavy chain and light chain (C1s-presenting autoantibodies) from patients with systemic lupus erythematosus (SLE), have been found to stimulate C1s enzymatic activities. This is due to acceleration of the proteolytic hydrolysis of the synthetic substrate C1-1 by C1s, enhancement of the complex formation of C1s with its natural pseudosubstrate, C1 inhibitor (C1 inh), and promotion of proteolytic activation of its natural substrate, C4. Seven of fifteen samples from patients with SLE were found to contain such autoantibodies. The hydrolysis of the synthetic substrate C1-1 catalyzed by C1s in 25 to 27 min in the presence of anti-C1s autoantibodies was equivalent to the hydrolysis of C1-1 catalyzed by C1s alone or C1s with control IgG from healthy sera in 110 min, approximately fourfold faster than the reaction in the absence of anti-C1s autoantibodies. Densitometry scanning data showed that the formation of the C1s-C1 inh complex in the presence of anti-C1s autoantibodies was three to four times greater than that with control IgG. It was also noticed that the autoantibodies convert almost all of the latent forms of C1s to an active form that binds to C1 inh. Another group of Western blots showed that C1s cleaved C4 α-chain three times faster in the presence of autoantibodies than of control IgG. It is likely that the overconsumption of complement components is common in the pathogenesis of tissue damage occurring in SLE.
Proteasomes Can Either Generate or Destroy MHC Class I Epitopes: Evidence for Nonproteasomal Epitope Generation in the Cytosol
Authors: Luckey Chance John , King Gina M. , Marto Jarrod A. , Venketeswaran Sunita , Maier Bernhard F. , Crotzer Victoria L. , Colella Teresa A. , Shabanowitz Jeffrey , Hunt Donald F. , Engelhard Victor H. 
Published: January 2023

AbstractProteasomes have been implicated in the production of the majority of peptides that associate with MHC class I molecules. We used two different proteasome inhibitors, the peptide aldehyde N-acetyl-l-leucyl-l-leucyl-l-norleucinal (LLnL) and the highly specific inhibitor lactacystin, to examine the role of proteasomes in generating peptide epitopes associated with HLA-A*0201. Neither LLnL nor lactacystin was able to completely block the expression of the HLA-A*0201. Furthermore, the effects of LLnL and lactacystin on the expression of different categories of specific epitopes, TAP independent vs TAP dependent and derived from either cytosolic or membrane proteins, were assessed. As predicted, presentation of two TAP-dependent epitopes was blocked by LLnL and lactacystin, while a TAP-independent epitope that is processed in the endoplasmic reticulum was unaffected by either inhibitor. Surprisingly, both LLnL and lactacystin increased rather than inhibited the expression of a cytosolically transcribed and TAP-dependent peptide from the influenza A virus M1 protein. Mass spectrometric analyses of in vitro proteasome digests of a synthetic 24 mer containing this epitope revealed no digestion products of any length that included the intact epitope. Instead, the major species resulted from cleavage sites within the epitope. Although cleavage at these sites was inhibitable by LLnL and lactacystin, epitope-containing species were still not produced. We conclude that proteasomes may in some cases actually destroy epitopes that would otherwise be destined for presentation by class I molecules. These results suggest that some epitopes are generated by nonproteasomal proteases in the cytosol.
A Partially Modified Retro-Inverso Pseudopeptide Modulates the Cytokine Profile of CTL Specific for an Influenza Virus Epitope
Authors: Ostankovitch Marina , Guichard Gilles , Connan Francine , Muller Sylviane , Chaboissier Aude , Hoebeke Johan , Choppin Jeannine , Briand Jean-Paul , Guillet Jean-Gérard 
Published: January 2023

Abstract
There is considerable evidence that peptides corresponding to MHC class I-restricted epitopes can be used as immunogens or immunomodulators. Pseudopeptides containing isosteric replacements of the amide bond provide more stable analogues, which may even have enhanced biologic activity. But there have been very few studies on the use of pseudopeptides to initiate or modulate the cellular immune response. This study describes the immunogenicity of a partially modified retro-inverso pseudopeptide of an influenza virus epitope and shows that this pseudopeptide modulates the cytokine profile expressed by CD8+CTL generated from primed precursors. Moreover, the pseudopeptide is much more efficient at low concentration than the wild-type epitope to stimulate IFN-γ secretion by CD8+ T effector cells. These results are analyzed with reference to changes in the conformation of the MHC molecule/peptide complex deduced from molecular modeling. The findings support the idea that partially modified retro-inverso analogues can be used as altered peptide ligands to enhance the stimulation of natural epitope-specific CTL and to modify their functional properties. Hence, pseudopeptide ligands might be promising tools for use in immunotherapy.