bluuebunny/crossref_metadata_2025 huggingface dataset:
B7 Requirements for Primary and Secondary Protein- and Polysaccharide-Specific Ig Isotype Responses toStreptococcus pneumoniae
Authors: Wu Zheng-Qi , Khan Abdul Q. , Shen Yi , Schartman Jerome , Peach Robert , Lees Andrew , Mond James J. , Gause William C. , Snapper Clifford M.
Published: April 2014

The requirements for B7 costimulation during an in vivo humoral response to an intact extracellular bacteria have not been reported. In this study we immunized mice with Streptococcus pneumoniae (R36A) to determine the B7 requirements for induction of Ig, specific for two determinants on R36A, the phosphorylcholine (PC) determinant of C-polysaccharide and pneumococcal surface protein A (PspA). We show that the primary anti-PspA response, the development of PspA-specific memory, and the induction of the secondary anti-PspA response in primed mice were completely dependent upon B7 costimulation. Of note, costimulation was required only briefly after the secondary immunization compared with after the primary immunization for optimal induction of Ig. Blockade of B7 costimulation at the time of secondary immunization also completely abrogated the established state of memory, but did not induce tolerance. In contrast to the anti-PspA response, the primary anti-PC response involved only a very short period of B7 costimulation. Whereas B7-2 alone was required for induction of the primary anti-PspA and anti-PC responses, a redundant role for B7-1 and B7-2 was noted for the PspA-specific secondary response. CTLA4Ig blocked both the anti-PC and anti-PspA responses equally well over a wide range of bacterial doses. These studies demonstrate a critical, but variable, role for B7-dependent costimulation during an Ig response to an extracellular bacteria.
Genetic Characterization of Strain Differences in the Ability to Mediate CD40/CD28-Independent Rejection of Skin Allografts
Authors: Williams Matthew A. , Trambley Joel , Ha Jongwon , Adams Andrew B. , Durham Megan M. , Rees Phyllis , Cowan Shannon R. , Pearson Thomas C. , Larsen Christian P.
Published: April 2014

Simultaneous blockade of the CD40 and CD28 T cell costimulatory pathways effectively promotes skin allograft survival in C3H/HeJ mice, extending median survival times (MSTs) beyond 100 days. This strategy is markedly less effective in C57BL/6 mice, with MSTs ranging between 20 and 30 days. In this study, we investigate the underlying genetic causes of these distinct phenotypes. Using H-2 congenic mice, we show that the genetic basis for the varied responses between these two strains is independent of the H-2 locus and T cell precursor frequency. C57BL/6 mice treated with costimulation blockade are able to generate allospecific CTL- and IFN-γ-producing T cells within 3–4 wk posttransplant, whereas mice with a C3H background generate neither CTL- nor IFN-γ-producing cells. Thus, differences appear to be in the generation of the immune response and not T cell homing. Strain differences in costimulation blockade-induced hyporesponsiveness persist in the absence of CD4+ T cells, implying a direct effect on CD8+ T cells. We demonstrate that genetic differences are important in cells of hemopoietic origin and that the costimulation blockade-resistant phenotype is dominant. Analysis of BXH recombinant inbred strains indicates that multiple loci contribute to the phenotype, and that the blockade resistance loci are preliminarily linked to 17 markers on four chromosomes. We conclude that strain variation in allograft MSTs following CD40/CD28 blockade results from the ability of CD8+ T cells in some strains to use alternative modes of costimulation to mount an effective alloresponse.
Reactivity and Regulatory Properties of Human Anti-Idiotypic Antibodies Induced by T Cell Vaccination
Authors: Hong Jian , Zang Ying C. Q. , Tejada-Simon Maria V. , Li Sufang , Rivera Victor M. , Killian James , Zhang Jingwu Z.
Published: April 2014

Immunization with irradiated autoreactive T cells (T cell vaccination) induces anti-idiotypic T cell responses that preferentially recognize complementarity-determining region 3 sequences, contributing to clonal depletion of autoreactive T cells. However, it remains unknown whether T cell vaccination elicits anti-idiotypic humoral responses and whether the anti-idiotypic Abs play a similar role in the regulatory mechanism induced by T cell vaccination. In this study we examined the occurrence, the reactivity pattern, and the regulatory role of anti-idiotypic Abs elicited by T cell vaccination in patients with multiple sclerosis. We demonstrated for the first time that B cells producing anti-idiotypic Abs could be isolated from vaccinated patients. These EBV-transformed B cell lines were selected for specific reactivity to a 20-mer TCR peptide incorporating a common complementarity-determining region 3 sequence of the immunizing T cell clones. The resulting anti-idiotypic Abs were found to react with the original immunizing T cell clones and exhibit an inhibitory effect on their proliferation. The findings suggest that anti-idiotypic Ab responses can be induced by T cell vaccination in humans and that their regulatory properties are likely to contribute to the suppression of myelin basic protein-reactive T cells in vaccinated patients. The study has important implications in our understanding of the regulatory role of the anti-idiotypic humoral responses induced by T cell vaccination.
Evidence for Protein Kinase C-Dependent and -Independent Activation of Mitogen-Activated Protein Kinase in T Cells: Potential Role of Additional Diacylglycerol Binding Proteins
Authors: Puente Lawrence G. , Stone James C. , Ostergaard Hanne L.
Published: April 2014

Activation of mitogen-activated protein kinases (MAPK) is a critical signal transduction event for CTL activation, but the signaling mechanisms responsible are not fully characterized. Protein kinase C (PKC) is thought to contribute to MAPK activation following TCR stimulation. We have found that dependence on PKC varies with the method used to stimulate the T cells. Extracellular signal-regulated kinase (ERK) activation in CTL stimulated with soluble cross-linked anti-CD3 is completely inhibited by the PKC inhibitor bisindolylmaleimide (BIM). In contrast, only the later time points in the course of ERK activation are sensitive to BIM when CTL are stimulated with immobilized anti-CD3, a condition that stimulates CTL degranulation. Surprisingly, MAPK activation in response to immobilized anti-CD3 is strongly inhibited at all time points by the diacylglycerol (DAG)-binding domain inhibitor calphostin C implicating the contribution of a DAG-dependent but PKC-independent pathway in the activation of ERK in CTL clones. Chronic exposure to phorbol ester down-regulates the expression of DAG-responsive PKC isoforms; however, this treatment of CTL clones does not inhibit anti-CD3-induced activation of MAPK. Phorbol ester-treated cells have reduced expression of several isoforms of PKC but still express the recently described DAG-binding Ras guanylnucleotide-releasing protein. These results indicate that the late phase of MAPK activation in CTL clones in response to immobilized anti-CD3 stimulation requires PKC while the early phase requires a DAG-dependent, BIM-resistant component.
The c-Abl Tyrosine Kinase Is Regulated Downstream of the B Cell Antigen Receptor and Interacts with CD19
Authors: Zipfel Patricia A. , Grove Matthew , Blackburn Kevin , Fujimoto Manabu , Tedder Thomas F. , Pendergast Ann Marie
Published: April 2014

c-Abl is a nonreceptor tyrosine kinase that we have recently linked to growth factor receptor signaling. The c-Abl kinase is ubiquitously expressed and localizes to the cytoplasm, plasma membrane, cytoskeleton, and nucleus. Thus, c-Abl may regulate signaling processes in multiple subcellular compartments. Targeted deletion or mutation of c-Abl in mice results in a variety of phenotypes, including splenic and thymic atrophy and lymphopenia. Additionally, lymphocytes isolated from specific compartments of c-Abl mutant mice have reduced responses to a variety of stimuli and an increased susceptibility to apoptosis following growth factor deprivation. Despite these observations, little is known regarding the signaling mechanisms responsible for these phenotypes. We report here that splenic B cells from c-Abl-deficient mice are hyporesponsive to the proliferative effects of B cell Ag receptor (BCR) stimulation. The c-Abl kinase activity and protein levels are elevated in the cytosol following activation of the BCR in B cell lines. We show that c-Abl associates with and phosphorylates the BCR coreceptor CD19, and that c-Abl and CD19 colocalize in lipid membrane rafts. These data suggest a role for c-Abl in the regulation of B cell proliferation downstream of the BCR, possibly through interactions with CD19.
Expression and Function of IL-12 and IL-18 Receptors on Human Tonsillar B Cells
Authors: Airoldi Irma , Gri Giorgia , Marshall Jason D. , Corcione Anna , Facchetti Paola , Guglielmino Roberta , Trinchieri Giorgio , Pistoia Vito
Published: April 2014

IL-12 activates murine and human B cells, but little information is available as to the expression and function of IL-12R on human B lymphocytes. Here we show that the latter cells, freshly isolated from human tonsils, expressed the transcripts of both β1 and β2 chains of IL-12R and that β2 chain mRNA was selectively increased (4- to 5-fold) by incubation with Staphylococcus aureus Cowan I bacteria or IL-12. B cell stimulation with IL-12 induced de novo expression of the transcripts of the two chains of IL-18R, i.e., IL-1 receptor-related protein and accessory protein-like. Functional studies showed that both IL-12 and IL-18 signaled to B cells through the NF-κB pathway. In the case of IL-12, no involvement of STAT transcription factors, and in particular of STAT-4, was detected. c-rel and p50 were identified as the members of NF-κB family involved in IL-12-mediated signal transduction to B cells. IL-12 and IL-18 synergized in the induction of IFN-γ production by tonsillar B cells, but not in the stimulation of B cell differentiation, although either cytokine promoted IgM secretion in culture supernatants. Finally, naive but not germinal center or memory, tonsillar B cells were identified as the exclusive IL-12 targets in terms of induction of NF-κB activation and of IFN-γ production.
CpG DNA Induces Maturation of Dendritic Cells with Distinct Effects on Nascent and Recycling MHC-II Antigen-Processing Mechanisms
Authors: Askew David , Chu Rose S. , Krieg Arthur M. , Harding Clifford V.
Published: April 2014

Murine bone marrow cultured with GM-CSF produced dendritic cells (DCs) expressing MHC class II (MHC-II) but little CD40, CD80, or CD86. Oligodeoxynucleotides (ODN) containing CpG motifs enhanced DC maturation, increased MHC-II expression, and induced high levels of CD40, CD80, and CD86. When added with Ag to DCs for 24 h, CpG ODN enhanced Ag processing, and the half-life of peptide:MHC-II complexes was increased. However, Ag processing was only transiently enhanced, and exposure of DCs to CpG ODN for 48 h blocked processing of hen egg lysozyme (HEL) to HEL48–61:I-Ak complexes. Processing of this epitope required newly synthesized MHC-II and was blocked by brefeldin A (BFA), suggesting that reduced MHC-II synthesis could explain decreased processing. Real-time quantitative PCR confirmed that CpG ODN decreased I-Aβk mRNA in DCs. In contrast, RNase42–56:I-Ak complexes were generated via a different processing mechanism that involved recycling MHC-II and was partially resistant to BFA. Processing of RNase42–56:I-Ak persisted, although at reduced levels, after CpG-induced maturation of DCs, and this residual processing by mature DCs was completely resistant to BFA. Changes in endocytosis, which was transiently enhanced and subsequently suppressed by CpG ODN, may affect Ag processing by both nascent and recycling MHC-II mechanisms. In summary, CpG ODN induce DC maturation, transiently increase Ag processing, and increase the half-life of peptide-MHC-II complexes to sustain subsequent presentation. Processing mechanisms that require nascent MHC-II are subsequently lost, but those that use recycling MHC-II persist even in fully mature DCs.
Negative Regulation of CD8+ T Cell Function by the IFN-Induced and Double-Stranded RNA-Activated Kinase PKR
Authors: Kadereit Suzanne , Xu Hui , Engeman Tara M. , Yang Yi-Li , Fairchild Robert L. , Williams Bryan R. G.
Published: April 2014

The IFN-induced and dsRNA-activated kinase (PKR) mediates the antiviral and antiproliferative effects of IFN-α and IFN-γ. Despite these findings, Pkr−/− mice have no overt immunological phenotype. Here we tested the role of PKR in cellular immunity by determining the induction and elicitation of contact hypersensitivity in Pkr−/− mice, a model of T cell-mediated immunity. When compared with wild type, the magnitude of contact hypersensitivity responses in Pkr−/− mice were 2-fold higher and of extended duration. This was also observed when naive recipients of immune CD8+ T cells from sensitized Pkr−/− and CD4+ T cells from sensitized wild-type Pkr+/+ or Pkr−/− mice were challenged with hapten, indicating a regulatory defect intrinsic to the CD8+ T cell population. Isolated lymph node T cells from Pkr−/− mice were hyperproliferative during Con A-mediated stimulation. These results implicate PKR for the first time in the growth control of mature T lymphocytes and give insight into the negative regulation of CD8+ T cell-mediated immune responses.
Receptor Revision in Peripheral T Cells Creates a Diverse Vβ Repertoire
Authors: McMahan Catherine J. , Fink Pamela J.
Published: April 2014

In Vβ5 transgenic mice, the age-dependent accumulation of Vβ5−CD4+ T cells expressing endogenous Vβ elements represents an exception to the rule of strict allelic exclusion at the TCRβ locus. The appearance of these cells is limited to the lymphoid periphery and is driven by a peripherally expressed tolerogen. Expression of the lymphoid-specific components of the recombinase machinery and the presence of recombination intermediates strongly suggest that TCR revision rescues tolerogen-reactive peripheral T cells from deletion. Here, we report that the appearance of Vβ5−CD4+ T cells is CD28-dependent. In addition, we find that the TCR repertoire of this unusual population of T cells in individual Vβ5 transgenic mice is surprisingly diverse, both at the level of surface protein and at the nucleotide level within a given family of V(D)Jβ rearrangements. This faithful recreation of the nontransgenic repertoire suggests that endogenous Vβ-expressing populations do not arise from expansion of an initially rare subset. Furthermore, the undersized N regions in revised TCR genes distinguish these sequences from those generated in the adult thymus. The diversity of the revised TCRs, the minimal mouse-to-mouse variation in the expressed endogenous Vβ repertoire, the atypical length of junctional sequences, and the CD28 dependence of the accumulation of Vβ5−CD4+ T cells all point to their extrathymic origin. Thus, tolerogen-driven receptor revision in peripheral T cells can expand the TCR repertoire extrathymically, thereby contributing to the flexibility of the immune repertoire.
Costimulation Light: Activation of CD4+ T Cells with CD80 or CD86 Rather Than Anti-CD28 Leads to a Th2 Cytokine Profile
Authors: Broeren Chris P. M. , Gray Gary S. , Carreno Beatriz M. , June Carl H.
Published: April 2014

To examine the role of CD28 and CTLA-4 in Th cell differentiation, we used a novel microsphere-based system to compare the effects of CD28 ligation by Ab or CD80/CD86. One set of beads was prepared by coating with anti-CD3 and anti-CD28 Ab. Another set of beads was prepared by immobilizing anti-CD3 and murine CD80-Ig fusion protein or murine CD86-Ig fusion protein on the beads. The three sets of beads were compared in their effects on the ability to activate and differentiate splenic CD4 T cells. When purified naive CD4+ cells were stimulated in vitro, robust proliferation of similar magnitude was induced by all three sets of beads. When cytokine secretion was examined, all bead preparations induced an equivalent accumulation of IL-2. In contrast, there was a marked difference in the cytokine secretion pattern of the Th2 cytokines IL-4, IL-10, and IL-13. The B7-Ig-stimulated cultures had high concentrations of Th2 cytokines, whereas there were low or undetectable concentrations in the anti-CD28-stimulated cultures. Addition of anti-CTLA-4 Fab augmented B7-mediated IL-4 secretion. These studies demonstrate that B7 is a critical and potent stimulator of Th2 differentiation, and that anti-CD28 prevents this effect.
A Role for CD21/CD35 and CD19 in Responses to Acute Septic Peritonitis: A Potential Mechanism for Mast Cell Activation
Authors: Gommerman Jennifer L. , Oh David Y. , Zhou Xiaoning , Tedder Thomas F. , Maurer Marcus , Galli Stephen J. , Carroll Michael C.
Published: April 2014

Although it is now appreciated that mast cell-mediated release of TNF-α is critical for resolution of acute septic peritonitis, questions remain as to how mast cells are activated upon peritoneal bacterial infection. Clues to how this may occur have been derived from earlier studies by Prodeus et al. in which complement proteins C3 and C4 were shown to be required for survival following cecal ligation and puncture (CLP), a model for acute septic peritonitis. To evaluate the mechanism for mast cell activation in the CLP model, complement receptor CD21/CD35-deficient mice (Cr2null) were examined in the present study. Along with CD19-deficient (CD19null) mice, these animals exhibit decreased survival following CLP compared with wild-type littermates. Injection of IgM before CLP does not change survival rates for Cr2null mice and only partially improves survival of CD19null mice, implicating CD21/CD35 and CD19 in mast cell activation. Interestingly, early TNF-α release is also impaired in Cr2null and CD19null animals, suggesting that these molecules directly affect mast cell activation. Cr2null and CD19null mice demonstrate an impairment in neutrophil recruitment and a corresponding increase in bacterial load. Examination of peritoneal mast cells by flow cytometry and confocal microscopy reveals the expression and colocalization of CD21/CD35 and CD19. Taken together, these findings suggest that the engagement of complement receptors CD21/CD35 along with CD19 on the mast cell surface by C3 fragments may be necessary for the full expression of mast cell activation in the CLP model.
Mapping the Ligand of the NK Inhibitory Receptor Ly49A on Living Cells
Authors: Chung Doo Hyun , Natarajan Kannan , Boyd Lisa F. , Tormo José , Mariuzza Roy A. , Yokoyama Wayne M. , Margulies David H.
Published: April 2014

We have used a recombinant, biotinylated form of the mouse NK cell inhibitory receptor, Ly49A, to visualize the expression of MHC class I (MHC-I) ligands on living lymphoid cells. A panel of murine strains, including MHC congenic lines, was examined. We detected binding of Ly49A to cells expressing H-2Dd, H-2Dk, and H-2Dp but not to those expressing other MHC molecules. Cells of the MHC-recombinant strain B10.PL (H-2u) not only bound Ly49A but also inhibited cytolysis by Ly49A+ effector cells, consistent with the correlation of in vitro binding and NK cell function. Binding of Ly49A to H-2Dd-bearing cells of different lymphoid tissues was proportional to the level of H-2Dd expression and was not related to the lineage of the cells examined. These binding results, interpreted in the context of amino acid sequence comparisons and the recently determined three-dimensional structure of the Ly49A/H-2Dd complex, suggest a role for amino acid residues at the amino-terminal end of the α1 helix of the MHC-I molecule for Ly49A interaction. This view is supported by a marked decrease in affinity of an H-2Dd mutant, I52 M, for Ly49A. Thus, allelic variation of MHC-I molecules controls measurable affinity for the NK inhibitory receptor Ly49A and explains differences in functional recognition in different mouse strains.
The Physical Association of Protein Kinase Cθ with a Lipid Raft-Associated Inhibitor of κB Factor Kinase (IKK) Complex Plays a Role in the Activation of the NF-κB Cascade by TCR and CD28
Authors: Khoshnan Ali , Bae David , Tindell Charles A. , Nel Andre E.
Published: April 2014

We investigated the role of protein kinase C θ (PKCθ) in the activation of the NF-κB cascade in primary human CD4+ lymphocytes. Among six or so PKC isoforms expressed in T cells, only PKCθ participates in the assembly of the supramolecular activation clusters at the contact site of the TCR with Ag. Signaling via both the TCR and CD28 is required for optimal activation of the multisubunit IκB kinase (IKK) complex in primary human T lymphocytes; this activation could be inhibited by a Ca2+-independent PKC isoform inhibitor, rottlerin. Moreover, endogenous PKCθ physically associates with activated IKK complexes in CD3/CD28-costimulated primary CD4+ T cells. The same set of stimuli also induced relocation of endogenous PKCθ and IKKs to a GM1 ganglioside-enriched, detergent-insoluble membrane compartment in primary T cells. IKKs recruited to these lipid rafts were capable of phosphorylating a recombinant IκBα sustrate. Confocal microscopy further demonstrated that exogenously expressed PKCθ and IKKβ colocalize in the membrane of CD3/CD28-costimulated Jurkat T cells. Constitutively active but not kinase-inactive PKCθ activated IKKβ in Jurkat T cells. Expression of dominant-active PKCθ also had stimulatory effects on the CD28 response element of the IL-2 promoter. Taken together, these data show that the activation of PKCθ by the TCR and CD28 plays an important role in the assembly and activation of IKK complexes in the T cell membrane.
Peroxisome Proliferator Activator Receptor-γ Agonists and 15-Deoxy-Δ12,1412,14-PGJ2 Induce Apoptosis in Normal and Malignant B-Lineage Cells
Authors: Padilla Josué , Kaur Kuljeet , Cao H. James , Smith Terry J. , Phipps Richard P.
Published: April 2014

The research described herein evaluates the expression and functional significance of peroxisome proliferator activator receptor-γ (PPAR-γ) on B-lineage cells. Normal mouse B cells and a variety of B lymphoma cells reflective of stages of B cell differentiation (e.g., 70Z/3, CH31, WEHI-231, CH12, and J558) express PPAR-γ mRNA and, by Western blot analysis, the 67-kDa PPAR-γ protein. 15-Deoxy-Δ12,14-PGJ2 (15d-PGJ2), a PPAR-γ agonist, has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [3H]thymidine and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays. Only PPAR-γ agonists (thiazolidinediones), and not PPAR-α agonists, mimicked the effect of 15d-PGJ2 on B-lineage cells, indicating that the mechanism by which 15d-PGJ2 negatively affects B-lineage cells involves in part PPAR-γ. The mechanism by which PPAR-γ agonists induce cytotoxicity is via apoptosis, as shown by annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay. Interestingly, addition of PGF2α, which was not known to affect lymphocytes, dramatically attenuated the deleterious effects of PPAR-γ agonists on B lymphomas. Surprisingly, 15d-PGJ2 induced a massive increase in nuclear mitogen-activated protein kinase activation, and pretreatment with PGF2α blunted the mitogen-activated protein kinase activation. This is the first study evaluating PPAR-γ expression and its significance on B lymphocytes. PPAR-γ agonists may serve as a counterbalance to the stimulating effects of other PGs, namely PGE2, which promotes B cell differentiation. Finally, the use of PGs, such as 15d-PGJ2, and synthetic PPAR-γ agonists to induce apoptosis in B-lineage cells may lead to the development of novel therapies for fatal B lymphomas.
Crystal Structure of an Antibody Bound to an Immunodominant Peptide Epitope: Novel Features in Peptide-Antibody Recognition
Authors: Nair Deepak T. , Singh Kavita , Sahu Naresh , Rao Kanury V. S. , Salunke Dinakar M.
Published: April 2014

The crystal structure of Fab of an Ab PC283 complexed with its corresponding peptide Ag, PS1 (HQLDPAFGANSTNPD), derived from the hepatitis B virus surface Ag was determined. The PS1 stretch Gln2P to Phe7P is present in the Ag binding site of the Ab, while the next three residues of the peptide are raised above the binding groove. The residues Ser11P, Thr12P, and Asn13P then loop back onto the Ag-binding site of the Ab. The last two residues, Pro14P and Asp15P, extend outside the binding site without forming any contacts with the Ab. The PC283-PS1 complex is among the few examples where the light chain complementarity-determining regions show more interactions than the heavy chain complementarity-determining regions, and a distal framework residue is involved in Ag binding. As seen from the crystal structure, most of the contacts between peptide and Ab are through the five residues, Leu3-Asp4-Pro5-Ala6-Phe7, of PS1. The paratope is predominantly hydrophobic with aromatic residues lining the binding pocket, although a salt bridge also contributes to stabilizing the Ag-Ab interaction. The molecular surface area buried upon PS1 binding is 756 Å2 for the peptide and 625 Å2 for the Fab, which is higher than what has been seen to date for Ab-peptide complexes. A comparison between PC283 structure and a homology model of its germline ancestor suggests that paratope optimization for PS1 occurs by improving both charge and shape complementarity.
The Transcription FactorBrightAssociates with Bruton’s Tyrosine Kinase, the Defective Protein in Immunodeficiency Disease
Authors: Webb Carol F. , Yamashita Yoshio , Ayers Neil , Evetts Seth , Paulin Yolene , Conley Mary Ellen , Smith Elizabeth A.
Published: April 2014

Binding of the transcription factor Bright to Ig heavy chain loci after B cell activation is associated with increased heavy chain transcription. We now report that Bright coprecipitates with Bruton’s tyrosine kinase (Btk), the defective enzyme in X-linked immunodeficiency disease (xid). Furthermore, we observed Btk in the nucleus of activated murine B cells, and mobility shift assays suggest that it is a component of the Bright DNA-binding complex. While Bright protein was synthesized in activated spleen cells from xid mice, it did not bind DNA or associate stably with Btk. These data suggest that deficiencies in Bright DNA-binding activity may contribute to the defects in Ig production seen in xid mice.
Identification of Fetal Liver Tyrosine Kinase 3 (Flt3) Ligand Domain Required for Receptor Binding and Function Using Naturally Occurring Ligand Isoforms
Authors: Mwangi Waithaka , Brown Wendy C. , Palmer Guy H.
Published: April 2014

We used a comparative approach to identify the fetal liver tyrosine kinase 3 (flt3) ligand structure required for binding and function. Two conserved bovine flt3 ligand isoforms, which differ in a defined region within the extracellular domain, were identified and shown to be uniformly transcribed in individuals with diverse MHC haplotypes. Notably, at the amino acid level, the extracellular domain of the bovine flt3 ligand isoform 1 is 81 and 72% identical with the extracellular domains of the human and murine flt3 ligands, respectively, whereas isoform-2 has a deletion within this domain. Bovine flt3 ligand isoform 1, but not 2, bound the human flt3 receptor and stimulated murine pro B cells transfected with the murine flt3 receptor. This retention of binding and function allowed definition of key residues by identifying sequences conserved among species. We have shown that a highly conserved, 18 aa sequence within the flt3 ligand extracellular domain is required for flt3 receptor binding and function. However, a peptide representing this sequence is insufficient for receptor binding as demonstrated by its failure to inhibit the bovine flt3 ligand isoform 1 binding to the human flt3 receptor. The requirement for flanking structure was confirmed by testing bovine flt3 ligand isoform 1 constructs truncated at specific residues outside the 18 aa sequence. Overall, the flt3 ligand structure required for function is markedly similar to that of the related hemopoietic growth factors, CSF-1 and steel factor. This definition of the required flt3 ligand structure will facilitate development of agonists to enhance dendritic cell recruitment for vaccines and immunotherapy.
The MurineIL-2Promoter Contains Distal Regulatory Elements Responsive to the Ah Receptor, a Member of the Evolutionarily Conserved bHLH-PAS Transcription Factor Family
Authors: Jeon Myung-Shin , Esser Charlotte
Published: April 2014

Signaling through the TCR and costimulatory signals primarily control transcription of the IL-2 gene in naive T cells. The minimal promoter necessary for this expression lies proximal, between −300 and the transcription start site. We had previously shown that activation of the arylhydrocarbon receptor (AHR), a member of the bHLH-PAS family of transcription factors, leads to increased mRNA expression of IL-2 in murine fetal thymocytes. The AHR is abundant in the thymus and may play a role for the development of the immune system. Moreover, its overactivation by chemicals such as dioxins leads to immunosuppression and thymic involution. Binding motifs for the liganded AHR can be identified in the distal region −1300 to −800 of the mouse IL-2 promoter. We show here that these DNA motifs, the so-called dioxin response elements, after binding to the liganded AHR are sufficient to transactivate luciferase expression in a reporter gene system. The IL-2 gene can be induced by the AHR also in thymocytes in vivo after injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent ligand of the AHR. The AHR mediates the IL-2 induction as shown with AHR-deficient mice. However, in spleen cells in vitro costimulation via the TCR is necessary for optimal IL-2 gene induction. Thus, the IL-2 promoter region contains novel distal regulatory elements that can be addressed by the AHR to induce IL-2 and can cooperate with the proximal promoter in this.
Conservation ofMhcClass III Region Synteny Between Zebrafish and Human as Determined by Radiation Hybrid Mapping
Authors: Sültmann Holger , Sato Akie , Murray Brent W. , Takezaki Naoko , Geisler Robert , Rauch Gerd-Jörg , Klein Jan
Published: April 2014

In the HLA, H2, and other mammalian Mhc, the class I and II loci are separated by the so-called class III region comprised of ∼60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the Mhc 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-Mhc that existed before the class I/II genes arose. The pre-Mhc may not have contained the complement and other class III genes involved in immune response.
Distinct T Cell Interactions with HLA Class II Tetramers Characterize a Spectrum of TCR Affinities in the Human Antigen-Specific T Cell Response
Authors: Reichstetter Sandra , Ettinger Ruth A. , Liu Andrew W. , Gebe John A. , Nepom Gerald T. , Kwok William W.
Published: April 2014

The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16369–379-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16369–379 peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16369–379 tetramer at either 23 or 37°C. Weak staining was also observed at 4°C. Clone 5 showed weaker staining compared with clone 48 at 37°C, and no staining was observed at 23°C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16369–379 complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.
Antibody Repertoire Development in Fetal and Neonatal Piglets. II. Characterization of Heavy Chain Complementarity-Determining Region 3 Diversity in the Developing Fetus
Authors: Butler J. E. , Weber P. , Sinkora M. , Sun J. , Ford S. J. , Christenson R. K.
Published: April 2014

Since the actual combinatorial diversity in the VH repertoire in fetal piglets represents <1% of the potential in mice and humans, we wondered whether 1) complementarity-determining region 3 (CDR3) diversity was also restricted; 2) CDR3 diversity changed with fetal age; and 3) to what extent CDR3 contributed to the preimmune VDJ repertoire. CDR3 spectratyping and sequence analyses of 213 CDR3s recovered from >30 fetal animals of different ages showed that >95% of VDJ diversity resulted from junctional diversity. Unlike sheep and cattle, somatic hypermutation does not contribute to the repertoire. These studies also revealed that 1) N region additions are as extensive in VDJ rearrangements recovered at 30 days as those in late term fetuses, suggesting that TdT is fully active at the onset of VDJ rearrangement; 2) nearly 90% of all rearrangement are in-frame until late gestation; 3) the oligoclonal CDR3 spectratype of 30-day fetal liver becomes polyclonal by 50 days, while this change occurs much later in spleen; 4) there is little evidence of individual variation in CDR3 spectratype or differences in spectratype among lymphoid tissues with the exception of the thymus; and 4) there is a tendency for usage of the most JH proximal DH segment (DHB) to decrease in older fetuses and for the longer DH segment to be trimmed to the same length as the shorter DH when used in CDR3. These findings suggest that in the fetal piglet, highly restricted combinatorial diversity and the lack of somatic mutation are compensated by early onset of TdT activity and other mechanisms that contribute to CDR3 junctional diversity.
Delineation of the Human Systemic Lupus Erythematosus Anti-Smith Antibody Response Using Phage-Display Combinatorial Libraries
Authors: del Rincon Inmaculada , Zeidel Maria , Rey Elena , Harley John B. , James Judith A. , Fischbach Michael , Sanz Iñaki
Published: April 2014

The anti-Smith (Sm) autoantibody response is highly specific for systemic lupus erythematosus and is predominantly targeted to the Sm-B/B′ and -D1 polypeptides. In all animal species thus far studied, anti-Sm Abs initially recognize proline-rich epitopes in the carboxyl terminus of the Sm-B/B′ protein and subsequently to multiple other epitopes in B/B′ and D. The absence of appropriate mAbs has limited our understanding of the genetic and structural basis of this autoimmune response. Using phage-display technology and lymphocytes from a systemic lupus erythematosus patient we have generated the first and only panel of human IgG anti-Sm mAbs thus far available. These Abs reproduced to a remarkable extent the serological reactivity of the patient. Epitope mapping and genetic studies revealed that the anti-Sm response is produced by distinct B cell clones with restricted epitope reactivity. All of the Abs in our study were exclusively encoded by different members of the VH4 gene family. On the aggregate, our results demonstrate that combinatorial libraries can recapitulate the immune repertoire of peripheral blood B memory cells and that epitope spreading appears to occur through the sequential recruitment of nonclonally related autoreactive B cell clones.
Activation of MHC Class I, II, and CD40 Gene Expression by Histone Deacetylase Inhibitors
Authors: Magner William J. , Kazim A. Latif , Stewart Carleton , Romano Michelle A. , Catalano Geoffrey , Grande Catherine , Keiser Nicholas , Santaniello Frank , Tomasi Thomas B.
Published: April 2014

Epigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-γ. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.
Factors Controlling the Trafficking and Processing of a Leader-Derived Peptide Presented by Qa-1
Authors: Bai Ailin , Aldrich Carla J. , Forman James
Published: April 2014

Many leader-derived peptides require TAP for presentation by class I molecules. This TAP dependence can either be ascribed to the inability of proteases resident in the endoplasmic reticulum (ER) to trim leader peptide precursors into the appropriate epitope or the failure of a portion of the leader segment to gain access to the lumen of the ER. Using the Qa-1 binding epitope, Qdm derived from a class Ia leader as a model, we show that many cell types lack ER protease activity to trim this peptide at its C terminus. However, both T1 and T2 cells contain appropriate protease activity to process the full length Dd leader (DL) when introduced into the ER lumen. Nevertheless, both T1 cells treated with the TAP inhibitor ICP47 and TAP− T2 cells fail to present this epitope from either the intact Dd molecule or a minigene encoding the DL. This indicates that the portion of the leader containing Qdm does not gain access to the ER. However, changing the Arg at P7 of the DL to a Cys can alter its trafficking and allows for TAP-independent presentation of the Qdm epitope.
HIV-1 Tat Represses Transcription from the Mannose Receptor Promoter
Authors: Caldwell Robert L. , Egan Brian S. , Shepherd Virginia L.
Published: April 2014

The mannose receptor is expressed on mature macrophages and immature dendritic cells, and functions to mediate phagocytosis of pathogens and capture of Ags for delivery to MHC class II-containing intracellular compartments. It has been previously reported that HIV-1-infected macrophages have reduced functions associated with the mannose receptor, including impaired Pneumocystis carinii phagocytosis and mannosylated albumin uptake. Several HIV-1-derived proteins including the Tat protein have been shown to transcriptionally repress host cell genes. The present study was undertaken to define the role of the HIV-1-derived protein Tat in HIV-mediated mannose receptor down-regulation. Cotransfection of the human macrophage cell line U937 with a Tat expression vector and a mannose receptor promoter-luciferase reporter construct resulted in down-regulation of mannose receptor promoter activity. This repression was targeted to the basal promoter. Expression of either one- or two-exon Tat resulted in decreased promoter activity. The addition of the transactivation response element (TAR) sequence enhanced the Tat-mediated repression. Down-regulation was also seen when transfected cells were treated with exogenously added Tat protein. These results are consistent with a mechanism whereby Tat reduces mannose receptor promoter activity by interfering with the host transcriptional initiation machinery, potentially resulting in decreased levels of surface mannose receptor available for Ag or pathogen capture.
Two Different Epitopes of the Signal Transducer gp130 Sequentially Cooperate on IL-6-Induced Receptor Activation
Authors: Pflanz Stefan , Kurth Ingo , Grötzinger Joachim , Heinrich Peter C. , Müller-Newen Gerhard
Published: April 2014

Cytokines are key mediators for the regulation of hemopoiesis and the coordination of immune responses. They exert their various functions through activation of specific cell surface receptors, thereby initiating intracellular signal transduction cascades which lead to defined cellular responses. As the common signal-transducing receptor subunit of at least seven different cytokines, gp130 is an important member of the family of hemopoietic cytokine receptors which are characterized by the presence of at least one cytokine-binding module. Mutants of gp130 that either lack the Ig-like domain D1 (ΔD1) or contain a distinct mutation (F191E) within the cytokine-binding module have been shown to be severely impaired with respect to IL-6 induced signal transduction. After cotransfection of COS-7 cells with a combination of both inactive gp130 mutants, signal transduction in response to IL-6 is restored. Whereas cells transfected with ΔD1 do not bind IL-6/sIL-6R complexes, cells transfected with the F191E mutant bind IL-6/sIL-6R with low affinity. Combination of ΔD1 and F191E, however, leads to high-affinity ligand binding. These data suggest that two different gp130 epitopes, one on each receptor chain, sequentially cooperate in asymmetrical binding of IL-6/IL-6R in a tetrameric signaling complex. On the basis of our data, a model for the mechanism of IL-6-induced gp130 activation is proposed.
Fab Chains As an Efficient Heterodimerization Scaffold for the Production of Recombinant Bispecific and Trispecific Antibody Derivatives
Authors: Schoonjans Reinhilde , Willems An , Schoonooghe Steve , Fiers Walter , Grooten Johan , Mertens Nico
Published: April 2014

Due to their multispecificity and versatility, bispecific Abs (BsAbs) are promising therapeutic tools in tomorrow’s medicine. Especially intermediate-sized BsAbs that combine body retention with tissue penetration are valuable for therapy but necessitate expression systems that favor heterodimerization of the binding sites for large-scale application. To identify heterodimerization domains to which single-chain variable fragments (scFv) can be fused, we compared the efficiency of heterodimerization of CL and CH1 constant domains with complete L and Fd chains in mammalian cells. We found that the isolated CL:CH1 domain interaction was inefficient for secretion of heterodimers. However, when the complete L and Fd chains were used, secretion of L:Fd heterodimers was highly successful. Because these Fab chains contribute a binding moiety, C-terminal fusion of a scFv molecule to the L and/or Fd chains generated BsAbs or trispecific Abs (TsAbs) of intermediate size (75–100 kDa). These disulfide-stabilized bispecific Fab-scFv (“bibody”) and trispecific Fab-(scFv)2 (“tribody”) heterodimers represent up to 90% of all secreted Ab fragments in the mammalian expression system and possess fully functional binding moieties. Furthermore, both molecules recruit and activate T cells in a tumor cell-dependent way, whereby the trispecific derivative can exert this activity to two different tumor cells. Thus we propose the use of the disulfide-stabilized L:Fd heterodimer as an efficient platform for production of intermediate-sized BsAbs and TsAbs in mammalian expression systems.
Induced κ Receptor Editing Shows No Allelic Preference in a Mouse Pre-B Cell Line
Authors: Liu Xiangdong , Linden Michael , Van Ness Brian
Published: April 2014

B cell Ag receptor editing is a process that can change κ antigen recognition specificity of a B cell receptor through secondary gene rearrangements on the same allele. In this study we used a model mouse pre-B cell line (38B9) to examine factors that might affect allelic targeting of secondary rearrangements of the κ locus. We isolated clones that showed both productive and nonproductive rearrangements of one κ allele, while retaining the other κ allele in the germline configuration (κ+/κ° or κ−/κ°). In the absence of any selective pressures, subsequent rearrangement of the germline alleles occurred at the same frequency as secondary rearrangement of the productive or nonproductive rearranged alleles. Because 38B9 cells lack Ig heavy chains, we stably expressed μ heavy chain protein in 38B9 cells to determine whether heavy-light pairing might affect allelic targeting of secondary κ rearrangements. Although the expression of heavy chain was found to both pair with and stabilize κ protein in these cells, it had no effect on preferential targeting Vκ-Jκ receptor editing compared with rearrangement of a germline allele. These studies suggest that in the absence of selection to eliminate autoreactive Vκ-Jκ genes, there is no allelic preference for secondary rearrangement events in 38B9 cells.
Immunization with a Recombinant Stage-Regulated Surface Protein fromLeishmania donovaniInduces Protection Against Visceral Leishmaniasis
Authors: Stäger Simona , Smith Deborah F. , Kaye Paul M.
Published: April 2014

Vaccination against visceral leishmaniasis has received limited attention compared with cutaneous leishmaniasis, although the need for an effective vaccine against visceral leishmaniasis is pressing. In this study, we demonstrate for the first time that a recombinant stage-specific hydrophilic surface protein of Leishmania donovani, recombinant hydrophilic acylated surface protein B1 (HASPB1), is able to confer protection against experimental challenge. Protection induced by rHASPB1 does not require adjuvant and, unlike soluble Leishmania Ag + IL-12, extends to the control of parasite burden in the spleen, an organ in which parasites usually persist and are refractory to a broad range of immunological and chemotherapeutic interventions. Both immunohistochemistry (for IL-12p40) and enzyme-linked immunospot assay (for IL-12p70) indicate that immunization with rHASPB1 results in IL-12 production by dendritic cells, although an analysis of Ab isotype responses to rHASPB1 suggests that this response is not sufficient in magnitude to induce a polarized Th1 response. Although both vaccinated and control-infected mice have equivalent frequencies of rHASPB1-specific CD4+ T cells producing IFN-γ, vaccine-induced protection correlates with the presence of rHASPB1-specific, IFN-γ-producing CD8+ T cells. Thus, we have identified a novel vaccine candidate Ag for visceral leishmaniasis, which appears to operate via a mechanism similar to that previously associated with DNA vaccination.