bluuebunny/crossref_metadata_2025_split huggingface dataset:
Initiation and Limitation of Ly-49A NK Cell Receptor Acquisition by T Cell Factor-1
Authors: Ioannidis Vassilios , Kunz Béatrice , Tanamachi Dawn M. , Scarpellino Léonardo , Held Werner 
Published: April 2014

Abstract
The establishment of clonally variable expression of MHC class I-specific receptors by NK cells is not well understood. The Ly-49A receptor is used by ≈20% of NK cells, whereby most cells express either the maternal or paternal allele and few express simultaneously both alleles. We have previously shown that NK cells expressing Ly-49A were reduced or almost absent in mice harboring a single or no functional allele of the transcription factor T cell factor-1 (TCF-1), respectively. In this study, we show that enforced expression of TCF-1 in transgenic mice yields an expanded Ly-49A subset. Even though the frequencies of Ly-49A+ NK cells varied as a function of the TCF-1 dosage, the relative abundance of mono- and biallelic Ly-49A cells was maintained. Mono- and biallelic Ly-49A NK cells were also observed in mice expressing exclusively a transgenic TCF-1, i.e., expressing a fixed amount of TCF-1 in all NK cells. These findings suggest that Ly-49A acquisition is a stochastic event due to limiting TCF-1 availability, rather than the consequence of clonally variable expression of the endogenous TCF-1 locus. Efficient Ly-49A acquisition depended on the expression of a TCF-1 isoform, which included a domain known to associate with the TCF-1 coactivator β-catenin. Indeed, the proximal Ly-49A promoter was β-catenin responsive in reporter gene assays. We thus propose that Ly-49A receptor expression is induced from a single allele in occasional NK cells due to a limitation in the amount of a transcription factor complex requiring TCF-1.
Induction of CD4+ T Cell Apoptosis as a Consequence of Impaired Cytoskeletal Rearrangement in UVB-Irradiated Dendritic Cells
Authors: Wachter Tina , Averbeck Marco , Hara Hisamichi , Tesmann Jens P. , Simon Jan C. , Termeer Christian C. , Denfeld Ralf W. 
Published: April 2014

AbstractLow dose UVB irradiation of dendritic cells (DC) dose-dependently decreases their allostimulatory capacity and inhibits alloreactive T cell proliferation. The reduction of the stimulatory capacity is not associated with a perturbation of CD28 costimulation. To examine the underlying mechanism, cell cycle analysis of T cells from cocultures with UVB-irradiated DC (UVB-DC) was performed, revealing no cell cycle arrest, but an increased number of apoptotic T cells in sub-G0 phase. We confirmed T cells to undergo apoptosis after coincubation with UVB-DC by TUNEL staining and DNA laddering. To analyze whether T cell apoptosis requires the Fas/Fas ligand (FasL) pathway, MLRs were performed with Fas-, FasL-deficient, and wild-type DC and T cells. No differences were found on comparison of wild-type DC with Fas-/FasL-deficient DC or T cells. Likewise, addition of a neutralizing anti-TNF-α mAb to cocultures could not overcome inhibition of T cell proliferation by UVB-DC, excluding involvement of the TNF-α/TNF-αR pathway. FACS analysis of CD69 and CD25 revealed no up-regulation on T cells cocultured with UVB-DC, suggesting a perturbation of early T cell activation. Analysis of UVB-DC by confocal microscopy demonstrated impaired filamentous actin bundling, a process critical for T cell stimulation. To investigate the functional relevance of these observations, time lapse video microscopy was performed. Indeed, calcium signaling in CD4+ T cells was significantly diminished after interaction with UVB-DC. In conclusion, UVBR of DC impairs their cytoskeletal rearrangement and induces apoptosis in CD4+ T cells by disruption of early DC-T cell interaction, resulting in a reduced Ca2+ influx in T cells.
Regulatory Roles of IL-2 and IL-4 in H4/Inducible Costimulator Expression on Activated CD4+ T Cells During Th Cell Development
Authors: Yagi Junji , Arimura Yutaka , Dianzani Umberto , Uede Toshimitsu , Okamoto Toshihiro , Uchiyama Takehiko 
Published: April 2014

AbstractWe found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4+ T cells obtained from six different murine strains. BALB/c-activated CD4+ T cells expressed ∼10-fold more H4/ICOS on their surfaces and produced ∼10-fold more IL-4 upon restimulation than C57BL/6-activated CD4+ T cells. BALB/c naive CD4+ T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4+ T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4+ T cells strongly down-regulated both H4/ICOS expression on activated CD4+ T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4+ T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4+ T cells from C57BL/6 or IL-4−/− mice increased H4/ICOS expression on activated CD4+ T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4+ T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4+ T cells.
Innate Immunity Together with Duration of Antigen Persistence Regulate Effector T Cell Induction
Authors: Storni Tazio , Ruedl Christiane , Renner Wolfgang A. , Bachmann Martin F. 
Published: April 2014

Abstract
Proliferation of T cells is important for the expansion of specific T cell clones during immune responses. In addition, for the establishment of protective immunity against viruses, bacteria, and tumors, the expanded T cells must differentiate into effector T cells. Here we show that effector T cell generation is driven by activation of APCs and duration of antigenic stimulation. Adoptively transferred TCR-transgenic T cells extensively proliferated upon immunization. However, these T cells failed to differentiate into effector cells and died within 1 wk after immunization unless antigenic peptides persisted for &gt;1 day or were presented by activated APCs. The induction of protective immunity in a nontransgenic system was more stringent, since activation of APCs or prolonged Ag persistence alone was not sufficient to drive immunity. In contrast, Ag had to be presented for several days by activated APCs to trigger protective T cell responses. Thus, activation of APCs and duration of Ag presentation together regulate the induction of protective T cell responses.
Characterization of Transcriptional Regulation During Negative Selection In Vivo
Authors: DeRyckere Deborah , Mann Derrick L. , DeGregori James 
Published: April 2014

AbstractNegative selection is the process whereby immature thymocytes expressing TCRs with high affinity for self-peptide:MHC complexes are induced to undergo apoptosis. The transcriptional events that occur as a result of TCR signaling during negative selection are not well-characterized. Using oligonucleotide arrays, we have identified 33 genes that exhibit changes in RNA levels in CD4+CD8+ thymocytes during negative selection in vivo. Of 18 genes that have been further characterized, 13 are regulated in response to stimulation with Ag or anti-CD3 and anti-CD28 Abs ex vivo, indicating that these genes are regulated independently of activation of the peripheral immune system. These data also support the idea that anti-CD3/CD28-mediated thymocyte apoptosis is a valid model for negative selection in vivo. A detailed examination of the regulation of many of the identified genes in response to treatment with dexamethasone or gamma-radiation or in response to anti-CD3/anti-CD28 stimulation in the presence of pharmacological inhibitors of mitogen-activated protein kinase kinase kinase 1, p38 mitogen-activated protein kinase, phosphatidylinositol 3-kinase, calcineurin, and cyclin-dependent kinase 2 has facilitated the elucidation of a map of the transcriptional events that occur downstream of the TCR. These studies support a model whereby similar signal transduction pathways are activated by stimuli that induce positive and negative selection and are consistent with the idea that the balance between opposing proapoptotic and antiapoptotic pathways determines cell fate. The data presented in this study also suggest that calcineurin functions to amplify TCR signals by promoting sustained increases in the levels of specific transcripts.
Modular Organization of the Carboxyl-Terminal, Globular Head Region of Human C1q A, B, and C Chains
Authors: Kishore Uday , Gupta Sanjeev K. , Perdikoulis Michael V. , Kojouharova Mihaela S. , Urban Britta C. , Reid Kenneth B. M. 
Published: April 2014

AbstractThe first step in the activation of the classical complement pathway, by immune complexes, involves the binding of the globular heads of C1q to the Fc regions of aggregated IgG or IgM. Located C-terminal to the collagen region, each globular head is composed of the C-terminal halves of one A (ghA), one B (ghB), and one C chain (ghC). To dissect their structural and functional autonomy, we have expressed ghA, ghB, and ghC in Escherichia coli as soluble proteins linked to maltose-binding protein (MBP). The affinity-purified fusion proteins (MBP-ghA, -ghB, and -ghC) bound differentially to heat-aggregated IgG and IgM, and also to three known C1q-binding peptides, derived from HIV-1, HTLV-I, and β-amyloid. In the ELISAs, the MBP-ghA bound to heat-aggregated IgG and IgM as well as to the HIV-1 gp41 peptide; the MBP-ghB bound preferentially to IgG rather than IgM, in addition to binding β-amyloid peptide, whereas the MBP-ghC showed a preference for IgM and the HTLV-I gp21 peptide. Both MBP-ghA and MBP-ghB also inhibited C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. However, for IgM-coated erythrocytes, MBP-ghC was a better inhibitor of C1q than MBP-ghB. The recombinant forms of ghA, ghB, and ghC also bound specifically to apoptotic PBMCs. We conclude that the C1q globular head region is likely to have a modular organization, being composed of three structurally and functionally independent modules, which retains multivalency in the form of a heterotrimer. The heterotrimeric organization thus offers functional flexibility and versatility to the whole C1q molecule.
Functional Cooperation of Simian Virus 40 Promoter Factor 1 and CCAAT/Enhancer-Binding Protein β and δ in Lipopolysaccharide-Induced Gene Activation of IL-10 in Mouse Macrophages
Authors: Liu Yi-Wen , Tseng Hui-Ping , Chen Lei-Chin , Chen Ben-Kuen , Chang Wen-Chang 
Published: April 2014

Abstract
Previous studies have revealed that LPS can activate transcription of the IL-10 gene promoter through an SV40 promoter factor 1 (Sp1) binding site in mouse macrophage RAW264.7. In this study, we determined that, in addition to Sp1, C/EBPβ and δ were also involved in LPS-induced gene expression of IL-10. By transient transfection with 5′-deletion mutants of the IL-10 promoter, we found that there were two LPS-responsive elements in the promoter of the mouse IL-10 gene. Analysis of these two regions by gel shift assay suggested that Sp1 and C/EBPβ and δ were bound to these two regions, respectively. By site-directed mutagenesis, we found that disruption at both the Sp1 and C/EBP binding sites almost completely blocked the LPS response. By gel shift assay and Western blotting, we found that the DNA binding complex and protein expression of C/EBPβ and δ were increased by LPS treatment, but these results were not found for Sp1. Overexpression of C/EBPβ or C/EBPδ, respectively, activated the promoter of the IL-10 gene, and they were enhanced by LPS. Coimmunoprecipitation experiments in intact cells indicated that LPS stimulated interaction between Sp1 and C/EBPβ and δ. These results suggested that the interaction between Sp1 and C/EBPβ and δ induced by LPS cooperatively activated expression of the IL-10 gene. The increase of C/EBPβ and δ proteins and the enhancement of transactivation activity of C/EBPβ and δ by LPS treatment, at least in part, explain the activation of IL-10 gene expression.
Increase of TCR Vβ Accessibility within Eβ Regulatory Region Influences its Recombination Frequency But Not Allelic Exclusion
Authors: Senoo Makoto , Wang Lili , Suzuki Daisuke , Takeda Naoki , Shinkai Yoichi , Habu Sonoko 
Published: April 2014

AbstractSeventy percent of the murine TCRβ locus (475 kb) was deleted to generate a large deleted TCRβ (βLD) allele to investigate a possible linkage between germline transcription, recombination frequency, and allelic exclusion of the TCR Vβ genes. In these βLD/LD mice, the TCRβ gene locus contained only four Vβ genes at the 5′ side of the locus, and consequently, the Vβ10 gene was located in the original Dβ1-Jβ1cluster within the Eβ regulatory region. We showed that the frequency of recombination and expression of the Vβ genes are strongly biased to Vβ10 in these mutant mice even though the proximity of the other three 5′Vβ genes was also greatly shortened toward the Dβ-Jβ cluster and the Eβ enhancer. Accordingly, the germline transcription of the Vβ10 gene in βLD/LD mice was exceptionally enhanced in immature double negative thymocytes compared with that in wild-type mice. During double negative-to-double positive transition of thymocytes, the level of Vβ10 germline transcription was prominently increased in βLD/LD recombination activating gene 2-deficient mice receiving anti-CD3ε Ab in vivo. Interestingly, however, despite the increased accessibility of the Vβ10 gene in terms of transcription, allelic exclusion of this Vβ gene was strictly maintained in βLD/LD mice. These results provide strong evidence that increase of Vβ accessibility influences frequency but not allelic exclusion of the TCR Vβ rearrangement if the Vβ gene is located in the Eβ regulatory region.
Hyperconservation of theN-Formyl Peptide Binding Site ofM3: Evidence thatM3Is an Old Eutherian Molecule with Conserved Recognition of a Pathogen-Associated Molecular Pattern
Authors: Doyle C. Kuyler , Davis Beckley K. , Cook Richard G. , Rich Robert R. , Rodgers John R. 
Published: April 2014

AbstractThe mouse MHC class I-b molecule H2-M3 has unique specificity for N-formyl peptides, derived from bacteria (and mitochondria), and is thus a pathogen-associated molecular pattern recognition receptor (PRR). To test whether M3 was selected for this PRR function, we studied M3 sequences from diverse murid species of murine genera Mus, Rattus, Apodemus, Diplothrix, Hybomys, Mastomys, and Tokudaia and of sigmodontine genera Sigmodon and Peromyscus. We found that M3 is highly conserved, and the 10 residues coordinating the N-formyl group are almost invariant. The ratio of nonsynonymous and synonymous substitution rates suggests the Ag recognition site of M3, unlike the Ag recognition site of class I-a molecules, is under strong negative (purifying) selection and has been for at least 50–65 million years. Consistent with this, M3 α1α2 domains from Rattus norvegicus and Sigmodon hispidus and from the “null” allele H2-M3b specifically bound N-formyl peptides. The pattern of nucleotide substitution in M3 suggests M3 arose rapidly from murid I-a precursors by an evolutionary leap (“saltation”), perhaps involving intense selective pressure from bacterial pathogens. Alternatively, M3 arose more slowly but prior to the radiation of eutherian (placental) mammals. Older dates for the emergence of M3, and the accepted antiquity of CD1, suggest that primordial class I MHC molecules could have evolved originally as monomorphic PRR, presenting pathogen-associated molecular patterns. Such MHC PRR molecules could have been preadaptations for the evolution of acquired immunity during the early vertebrate radiation.
T Cell Antagonism is Functionally Uncoupled from the 21- and 23-kDa Tyrosine-Phosphorylated TCR ζ Subunits
Authors: Pitcher Lisa A. , Ohashi Pamela S. , van Oers Nicolai S. C. 
Published: April 2014

AbstractThe functional effects of altered peptide ligands on T cells is proposed to involve differential intracellular signaling mediated by the 21- and 23-kDa tyrosine-phosphorylated derivatives of the TCR ζ subunit (p21 and p23). To understand the functional contribution of p21 and p23 to T cell development and T cell antagonism, we generated selected TCR ζ transgenic mice maintained on the P14 αβ TCR transgenic line such that p23 or both p21 and p23 were selectively eliminated. Importantly, one line (YF1,2) retains the constitutively tyrosine-phosphorylated p21 in the complete absence of inducible p23. We determined that T cell development was uncoupled from p21 and/or p23. Using a series of agonist, weak agonist, and antagonist peptides, we analyzed the role of each of the phosphorylated forms of TCR ζ on T cell activation and antagonism. In this study, we report that the proliferative responses of αβ P14 T cells to agonist peptides and the inhibition of proliferation resulting from antagonist peptide treatments was functionally uncoupled from p21 and/or p23. These results suggest that the mechanism of T cell antagonism is independent of the two phosphorylated TCR ζ derivatives.
Editing of an Immunodominant Epitope of Glutamate Decarboxylase by HLA-DM
Authors: Lich John D. , Jayne Jennifer A. , Zhou Delu , Elliott John F. , Blum Janice S. 
Published: April 2014

AbstractHLA-DM stabilizes peptide-receptive class II αβ dimers and facilitates the capture of high affinity peptides, thus influencing the peptide repertoire presented by class II molecules. Variations in DM levels may therefore have a profound effect on the antigenic focus of T cell-mediated immune responses. Specifically, DM expression may influence susceptibility and resistance to autoimmune diseases. In this study the role of DM in HLA-DR4-restricted presentation of an insulin-dependent diabetes mellitus autoantigen, glutamate decarboxylase (GAD), was tested. Presentation of immunodominant GAD epitope 273–285 was regulated by endogenous DM levels in human B lymphoblasts. T cell responses to exogenous GAD as well as an endogenous cytoplasmic form of this Ag were significantly diminished with increasing cellular expression of DM. Epitope editing by DM was observed only using Ag and not small synthetic peptides, suggesting that this process occurred within endosomes. Results with cytoplasmic GAD also indicated that peptides from this compartment intersect class II proteins in endocytic vesicles where DM editing was facilitated. Changes in DM levels within APC may therefore influence the presentation of autoantigens and the development of autoimmune disorders such as type I diabetes.
Linker for Activation of T Cells, ζ-Associated Protein-70, and Src Homology 2 Domain-Containing Leukocyte Protein-76 are Required for TCR-Induced Microtubule-Organizing Center Polarization
Authors: Kuhné Michelle R. , Lin Joseph , Yablonski Deborah , Mollenauer Marianne N. , Ehrlich Lauren I. Richie , Huppa Johannes , Davis Mark M. , Weiss Arthur 
Published: April 2014

Abstract
Engagement of the T cell with Ag on an APC results in a series of immediate signaling events emanating from the stimulation of the TCR. These events include the induced phosphorylation of a number of cellular proteins with a subsequent increase in intracellular calcium and the restructuring of the microtubule and actin cytoskeleton within the T cell. This restructuring of the cytoskeleton culminates in the polarization of the T cell’s secretory apparatus toward the engaging APC, allowing the T cell to direct secretion of cytokines toward the appropriate APC. This polarization can be monitored by analyzing the position of the microtubule-organizing center (MTOC), as it moves toward the interface of the T cell and APC. The requirements for MTOC polarization were examined at a single-cell level by studying the interaction of a Jurkat cell line expressing a fluorescently labeled MTOC with Staphylococcal enterotoxin superantigen-bound Raji B cell line, which served as the APC. We found that repolarization of the MTOC substantially followed fluxes in calcium. We also used immobilized anti-TCR mAb and Jurkat signaling mutants, defective in TCR-induced calcium increases, to determine whether signaling components that are necessary for a calcium response also play a role in MTOC polarization. We found that ζ-associated protein-70 as well as its substrate adaptor proteins linker for activation of T cells and Src homology 2 domain-containing leukocyte protein-76 are required for MTOC polarization. Moreover, our studies revealed that a calcium-dependent event not requiring calcineurin or calcium/calmodulin-dependent kinase is required for TCR-induced polarization of the MTOC.
Stalk Region of β-Chain Enhances the Coreceptor Function of CD8
Authors: Wong Jenny S. , Wang Xiaosong , Witte Torsten , Nie Linghu , Carvou Nicolas , Kern Petra , Chang Hsiu-Ching 
Published: April 2014

AbstractCD8 glycoproteins are expressed as either αα homodimers or αβ heterodimers on the surface of T cells. CD8αβ is a more efficient coreceptor than the CD8αα for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8αβ is a better coreceptor than CD8αα, we engineered, expressed, and functionally tested a chimeric CD8α protein whose stalk region is replaced with that of CD8β. We found that the β stalk region enhances the coreceptor function of chimeric CD8αα to a level similar to that of CD8αβ. Surprisingly, the β stalk region also restored functional activity to an inactive CD8α variant, carrying an Ala mutation at Arg8 (R8A), to a level similar to that of wild-type CD8αβ. Using the R8A variant of CD8α, a panel of anti-CD8α Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8α interaction, we show that the introduction of the CD8β stalk leads to a different topology of the CD8α-MHCI complex without altering the overall structure of the Ig-like domain of CD8α or causing the MHCI to employ different residues to interact with the CD8α Ig domain. Our results show that the stalk region of CD8β is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.
Characterization of Pig-Tailed Macaque Classical MHC Class I Genes: Implications for MHC Evolution and Antigen Presentation in Macaques
Authors: Lafont Bernard A. P. , Buckler-White Alicia , Plishka Ron , Buckler Charles , Martin Malcolm A. 
Published: April 2014

AbstractMHC-dependent CD8+ T cell responses have been associated with control of viral replication and slower disease progression during lentiviral infections. Pig-tailed macaques (Macaca nemestrina) and rhesus monkeys (Macaca mulatta), two nonhuman primate species commonly used to model HIV infection, can exhibit distinct clinical courses after infection with different primate lentiviruses. As an initial step in assessing the role of MHC class I restricted immune responses to these infections, we have cloned and characterized classical MHC class I genes of pig-tailed macaques and have identified 19 MHC class I alleles (Mane) orthologous to rhesus macaque MHC-A, -B, and -I genes. Both Mane-A and Mane-B loci were found to be duplicated, and no MHC-C locus was detected. Pig-tailed and rhesus macaque MHC-A alleles form two groups, as defined by 14 polymorphisms affecting mainly their B peptide-binding pockets. Furthermore, an analysis of multiple pig-tailed monkeys revealed the existence of three MHC-A haplotypes. The distribution of these haplotypes in various Old World monkeys provides new insights about MHC-A evolution in nonhuman primates. An examination of B and F peptide-binding pockets in rhesus and pig-tailed macaques suggests that their MHC-B molecules present few common peptides to their respective CTLs.