bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoglobulin M Efficacy AgainstCryptococcus neoformans: Mechanism, Dose Dependence, and Prozone-Like Effects in Passive Protection Experiments
Authors: Taborda Carlos P. , Casadevall Arturo
Published: April 2014

The IgM mAbs 12A1 and 13F1 are protective and nonprotective, respectively, against lethal Cryptococcus neoformans infection in mice. To better understand the variables that contribute to IgM efficacy against C. neoformans, we studied the effects of inoculum size, route of infection, and Ab dose for each of these mAbs. mAb 13F1 did not prolong survival under any condition studied. mAb 12A1 prolonged survival after the administration of certain Ab doses after i.p. infection with defined inocula and promoted phagocytosis, agglutination, and the formation of inflammatory cell rings around yeast cells in vivo. Large Ab doses of mAb 12A1 resulted in either no protection or enhanced infection, consistent with a prozone-like effect. Investigation of this phenomenon revealed that the fungal cell was protected against microbicidal nitrogen-derived oxidants when large amounts of Ab were bound to the C. neoformans capsule. mAb 12A1 was opsonic in vitro for peritoneal, but not splenic or alveolar macrophages. In summary, our results indicate that IgM efficacy against C. neoformans is a function of the route of infection, inoculum, and Ab dose and is associated with its ability to promote opsonization, agglutination, and phagocytic ring formation in vivo. The occurrence of the prozone-like phenomenon implies that high Ab titers are not necessarily beneficial in assuring protection against certain pathogens and that caution should be exercised in using high Ab titer as a measure for vaccine efficacy.
Amelioration of Experimental Autoimmune Encephalomyelitis with Anti-OX40 Ligand Monoclonal Antibody: A Critical Role for OX40 Ligand in Migration, But Not Development, of Pathogenic T Cells
Authors: Nohara Chiyoko , Akiba Hisaya , Nakajima Atsuo , Inoue Atsushi , Koh Chang-Sung , Ohshima Hideo , Yagita Hideo , Mizuno Yoshikuni , Okumura Ko
Published: April 2014

OX40 (CD134) and its ligand (OX40L) have been implicated in T cell activation and migration. In this study, we examined the contribution of these molecules to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) by administering a neutralizing mAb against murine OX40L (RM134L) to proteolipid protein (139–151) peptide-induced EAE in SJL mice. Administration of RM134L effectively ameliorated the disease in both actively induced and adoptively transferred EAE models. Histological examination showed that the RM134L treatment greatly reduced mononuclear cell infiltration into the spinal cord. The RM134L treatment did not inhibit the development of pathogenic T cells, given that proliferative response and IFN-γ production by draining lymph node cells were not reduced or rather enhanced upon restimulation with proteolipid protein (139–151) in vitro, and these cells effectively transferred EAE to naive SJL mice. Flow cytometric analyses showed that the RM134L treatment inhibited the accumulation of OX40-expressing CD4+ T cells and the migration of adoptively transferred CD4+ T cells in the spinal cord. Immunohistochemical staining showed that OX40L was most prominently expressed on endothelial cells in the inflamed spinal cord. These results suggest that the OX40/OX40L interaction plays a critical role for the migration of pathogenic T cells into the CNS in the pathogenesis of EAE.
Antigen-Specific Mediated Suppression of β Cell Autoimmunity by Plasmid DNA Vaccination
Authors: Tisch Roland , Wang Bo , Weaver Donald J. , Liu Bo , Bui Thi , Arthos James , Serreze David V.
Published: April 2014

In this study, we have investigated the use of plasmid DNA (pDNA) vaccination to elicit Th2 effector cell function in an Ag-specific manner and in turn prevent insulin-dependent diabetes mellitus (IDDM) in nonobese diabetic (NOD) mice. pDNA recombinants were engineered encoding a secreted fusion protein consisting of a fragment of glutamic acid decarboxylase 65 (GAD65) linked to IgGFc, and IL-4. Intramuscular injection of pDNA encoding GAD65-IgGFc and IL-4 effectively prevented diabetes in NOD mice treated at early or late preclinical stages of IDDM. This protection was GAD65-specific since NOD mice immunized with pDNA encoding hen egg lysozyme-IgGFc and IL-4 continued to develop diabetes. Furthermore, disease prevention correlated with suppression of insulitis and induction of GAD65-specific regulatory Th2 cells. Importantly, GAD65-specific immune deviation was dependent on pDNA-encoded IL-4. In fact, GAD65-specific Th1 cell reactivity was significantly enhanced in animals immunized with pDNA encoding only GAD65-IgGFc. Finally, NOD.IL4null mice treated with pDNA encoding GAD65-IgGFc and IL-4 continued to develop diabetes, indicating that endogenous IL-4 was also required for disease prevention. These results demonstrate that pDNA vaccination is an effective strategy to elicit β cell-specific Th2 regulatory cell function for the purpose of preventing IDDM even at a late stage of disease development.
The Growth of the Very Large CD8+ T Cell Clones in Older Mice Is Controlled by Cytokines
Authors: Ku Chia-Chi , Kappler John , Marrack Philippa
Published: April 2014

Older humans and mice frequently contain very large clones of CD8+ T cells. In mice these cells are phenotypically very similar to memory CD8+ T cells. Like memory CD8+ T cells, most members of the clones are in continuous slow division, apparently independently of Ag stimulation. Proliferation of the CD8+ clonal T cells is inhibited in mice treated with Ab to the IL-2R β-chain that blocks signaling by either IL-2 or IL-15. However, inhibition of IL-2 increases the numbers of dividing clonal cells. Therefore, like normal memory CD8+ T cells, expansion of the clones is driven by IL-15 and inhibited by IL-2 and is probably limited by the amounts of IL-15 and IL-2 present in the host. Control by these two cytokines may account for the fact that, although the clones can be very large, they do not overwhelm or kill their hosts. Nevertheless the clonal cells compete successfully with normal memory CD8+ T cells for growth. Perhaps the clonal cells use IL-15 more effectively or are more resistant to the inhibitory effects of IL-2. Thus they might affect the immune response of their hosts by competing for factors that stimulate and inhibit normal CD8+ memory T cells.
Human Thymic Epithelial Cells Inhibit IL-15- and IL-2-Driven Differentiation of NK Cells from the Early Human Thymic Progenitors
Authors: Le Phong T. , Adams Kimberly L. , Zaya Ninef , Mathews Herbert L. , Storkus Walter J. , Ellis Thomas M.
Published: April 2014

T/NK progenitors are present in the thymus; however, the thymus predominantly promotes T cell development. In this study, we demonstrated that human thymic epithelial cells (TEC) inhibit NK cell development. Most ex vivo human thymocytes express CD1a, indicating that thymic progenitors are predominantly committed to the T cell lineage. In contrast, the CD1a−CD3−CD56+ NK population comprises only 0.2% (n = 7) of thymocytes. However, we observed increases in the percentage (20- to 25-fold) and absolute number (13- to 71-fold) of NK cells when thymocytes were cultured with mixtures of either IL-2, IL-7, and stem cell factor or IL-15, IL-7, and stem cell factor. TEC, when present in the cultures, inhibited the increases in the percentage (3- to 10-fold) and absolute number (3- to 25-fold) of NK cells. Furthermore, we show that TEC-derived soluble factors inhibit generation of NK-CFU and inhibit IL15- or IL2-driven NK cell differentiation from thymic CD34+ triple-negative thymocytes. The inhibitory activity was found to be associated with a 8,000- to 30,000 Da fraction. Thus, our data demonstrate that TEC inhibit NK cell development from T/NK CD34+ triple negative progenitors via soluble factor(s), suggesting that the human thymic microenvironment not only actively promotes T cell maturation but also controls the development of non-T lineage cells such as the NK lineage.
Activated p56lck Directs Maturation of Both CD4 and CD8 Single-Positive Thymocytes
Authors: Sohn Sue J. , Forbush Katherine A. , Pan Xiao Cun , Perlmutter Roger M.
Published: April 2014

p56lck is a protein tyrosine kinase expressed throughout T cell development. It associates noncovalently with the cytoplasmic domains of the CD4 and CD8 coreceptor molecules and has been implicated in TCR signaling in mature T cells. Its role in early thymocyte differentiation has been demonstrated in vivo, both by targeted gene disruption and by transgene expression. Previously, we showed that expression of a dominant-negative form of p56lck in double-positive thymocytes inhibits positive selection. We now demonstrate that expression of constitutively activated p56lck (p56lckF505) accelerates the transition from the double-positive to the single-positive stage. Importantly, p56lckF505 drives survival and lineage commitment of thymocytes in the absence of TCR engagement by appropriate MHC molecules. These results indicate that activation of p56lck constitutes an early step in conveying maturational signals after TCR ligation by a positively selecting ligand. Our study provides direct in vivo evidence for the role of p56lck in regulating TCR signaling.
NF-κB/Rel Participation in the Lymphokine-Dependent Proliferation of T Lymphoid Cells
Authors: Mora Ana L. , Youn Jeehee , Keegan Achsah D. , Boothby Mark
Published: April 2014

Proliferative responses of lymphoid cells to IL-2 and IL-4 depend on activation of the cells, but the mechanism(s) by which activation enhances cellular competence to respond to cytokines is not fully understood. The NF-κB/Rel family represents one signal transduction pathway induced during such activation. We show in this study that inhibition of NF-κB through the expression of an IκBα (inhibitory protein that dissociates from NF-κB) mutant refractory to signal-induced degradation (IκBα(ΔN)) interfered with the acquisition of competence to proliferate in response to IL-4 as well as IL-2. Thymocytes and T cells from IκBα(ΔN) transgenic mice expressed normal levels of IL-2R subunits. However, transgenic cells exhibited a dramatic defect in Stat5A activation treatment with IL-2, and a similar defect was observed for IL-4-induced Stat5. In contrast, T lymphoid cells with inhibition of NF-κB showed normal insulin receptor substrate-2 phosphorylation and only a modest decrease in Stat6 activation and insulin receptor substrate-1 phosphorylation after IL-4 stimulation. These results indicate that the NF-κB/Rel/IκBα system can regulate cytokine receptor capacitation through effects on the induction of downstream signaling by the Stat transcription factor family.
IFN-γ Regulation of Class II Transactivator Promoter IV in Macrophages and Microglia: Involvement of the Suppressors of Cytokine Signaling-1 Protein
Authors: O’Keefe George M. , Nguyen Vince T. , Ping Tang Li , Benveniste Etty N.
Published: April 2014

The discovery of the class II transactivator (CIITA) transcription factor, and its IFN-γ-activated promoter (promoter IV), have provided new opportunities to understand the molecular mechanisms of IFN-γ-induced class II MHC expression. Here, we investigated the molecular regulation of IFN-γ-induced murine CIITA promoter IV activity in microglia/macrophages. In the macrophage cell line RAW264.7, IFN-γ inducibility of CIITA promoter IV is dependent on an IFN-γ activation sequence (GAS) element and adjacent E-Box, and an IFN response factor (IRF) element, all within 196 bp of the transcription start site. In both RAW cells and the microglia cell line EOC20, two IFN-γ-activated transcription factors, STAT-1α and IRF-1, bind the GAS and IRF elements, respectively. The E-Box binds upstream stimulating factor-1 (USF-1), a constitutively expressed transcription factor. Functionally, the GAS, E-Box, and IRF elements are each essential for IFN-γ-induced CIITA promoter IV activity. The effects of the suppressors of cytokine signaling-1 (SOCS-1) protein on IFN-γ-induced CIITA and class II MHC expression were examined. Ectopic expression of SOCS-1 inhibits IFN-γ-induced activation of CIITA promoter IV and subsequent class II MHC protein expression. Interestingly, SOCS-1 inhibits the constitutive expression of STAT-1α and its IFN-γ-induced tyrosine phosphorylation and binding to the GAS element in CIITA promoter IV. As well, IFN-γ-induced expression of IRF-1 and its binding to the IRF element is inhibited. These results indicate that SOCS-1 may be responsible for attenuating IFN-γ-induced CIITA and class II MHC expression in macrophages.
Distinct CpG DNA and Polyinosinic-Polycytidylic Acid Double-Stranded RNA, Respectively, Stimulate CD11c− Type 2 Dendritic Cell Precursors and CD11c+ Dendritic Cells to Produce Type I IFN
Authors: Kadowaki Norimitsu , Antonenko Svetlana , Liu Yong-Jun
Published: April 2014

Two classes of nucleic acids, bacterial DNA containing unmethylated CpG motifs and dsRNA in viruses, induce the production of type I IFN that contributes to the immunostimulatory effects of these microbial molecules. Thus, it is important to determine which cells produce type I IFN in response to CpG DNA and dsRNA. CD4+CD11c− type 2 dendritic cell precursors (pre-DC2) were identified as the main producers of type I IFN in human blood in response to viruses. Here we asked whether pre-DC2 also produce type I IFN in response to CpG DNA and dsRNA. Oligodeoxynucleotides containing particular palindromic CpG motifs induced pre-DC2, but not CD11c+ blood DC or monocytes, to produce IFN-α. In contrast, a synthetic dsRNA, polyinosinic polycytidylic-acid, induced CD11c+ DC, but not pre-DC2 or monocytes, to produce IFN-αβ. These data indicate that CpG DNA and polyinosinic-polycytidylic acid stimulate different types of cells to produce type I IFN and that it is important to select oligodeoxynucleotides containing particular CpG motifs to induce pre-DC2 to produce type I IFN, which may play a key role in the strong adjuvant effects of CpG DNA.
Peripheral Deletion After Bone Marrow Transplantation with Costimulatory Blockade Has Features of Both Activation-Induced Cell Death and Passive Cell Death
Authors: Wekerle Thomas , Kurtz Josef , Sayegh Mohamed H. , Ito Hiroshi , Wells Andrew D. , Bensinger Steven , Shaffer Juanita , Turka Laurence A. , Sykes Megan
Published: April 2014

Two major pathways of death of previously activated T cells have been described: activation-induced cell death can be triggered by restimulating activated T cells with high concentrations of Ag, is Fas-dependent, is not influenced by proteins of the Bcl family, and is blocked by cyclosporin A; in contrast, passive cell death is induced by the withdrawal of growth factors and activation stimuli, is Fas-independent, and is blocked by Bcl family proteins. We examined the role of these two forms of cell death in the peripheral deletion of donor-reactive host T cells after allogeneic bone marrow transplantation and costimulatory blockade with anti-CD154 plus CTLA4Ig in two murine models. The substantial decline in donor-reactive CD4 cells seen in wild-type recipients 1 wk after bone marrow transplantation with costimulatory blockade was largely inhibited in Fas-deficient recipients and in Bcl-xL-transgenic recipients. We observed these effects both in a model involving low-dose total body irradiation and a conventional dose of bone marrow, and in a radiation-free regimen using high-dose bone marrow transplantation. Furthermore, cyclosporin A did not completely block the deletion of donor-reactive CD4+ T cells in recipients of bone marrow transplantation with costimulatory blockade. Thus, the deletion of donor-reactive T cells occurring early after bone marrow transplantation with costimulatory blockade has features of both activation-induced cell death and passive cell death. Furthermore, these in vivo data demonstrate for the first time the significance of in vitro results indicating that proteins of the Bcl family can prevent Fas-mediated apoptosis under certain circumstances.
Lysophosphatidic Acid Receptor-Selective Effects on Jurkat T Cell Migration Through a Matrigel Model Basement Membrane
Authors: Zheng Yuhua , Kong Yvonne , Goetzl Edward J.
Published: April 2014

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) from platelets and mononuclear phagocytes mediate T cell functions through endothelial differentiation gene-encoded G protein-coupled receptors (Edg Rs) specific for LPA (Edg-2, -4, and -7) or S1P (Edg-1, -3, -5, -6, and -8). Jurkat leukemic T cells with the SV40 virus large T Ag (Jurkat-T cells) express Edg-3>-2>-4 Rs, as assessed by RT-semiquantitative PCR and Western blots with anti-Edg R mAbs. Jurkat-T cells expressing predominantly Edg-2 R (Jurkat-T-2 cells) and Edg-4 R (Jurkat-T-4 cells) were developed by cotransfection with the respective sense plasmids and a mixture of antisense plasmids for the other Edg Rs, and hygromycin selection. Migration of Jurkat-T-4 cells, but not Jurkat-T-2 cells, through a layer of Matrigel on a 5-um pore polycarbonate filter was stimulated up to 5-fold by 10−9 to 10−6 M LPA and by 30–300 ng/ml of anti-Edg-4 R Ab, but not anti-Edg-2 R Ab. LPA and anti-Edg-4 R Ab also enhanced by up to 4-fold the expression of matrix metalloproteinase by Jurkat-T-4 cells, but not Jurkat-T-2 cells, as assessed by cleavage of [3H]-type IV human collagen in the Matrigel. Enhancement of matrix metalloproteinase-dependent trans-Matrigel migration of Jurkat-T cells by the chemokine RANTES was suppressed by anti-Edg-2 R Abs, but was stimulated by anti-Edg-4 R Abs. The opposite effects of Edg-2 and Edg-4 LPA receptors on trans-Matrigel migration and some other T cell functions provide receptor-selective mechanisms for regulation of T cell recruitment and immune contributions.
Ly-49W, an Activating Receptor of Nonobese Diabetic Mice With Close Homology to the Inhibitory Receptor Ly-49G, Recognizes H-2Dk and H-2Dd
Authors: Silver Elizabeth T. , Gong Dong-Er , Hazes Bart , Kane Kevin P.
Published: April 2014

The diversity and ligand specificity of activating Ly-49 receptors expressed by murine NK cells are largely unknown. We cloned a new Ly-49-activating receptor, expressed by NK cells of the nonobese diabetic mouse strain, which we have designated Ly-49W. Ly-49W is highly related to the known inhibitory receptor Ly-49G in its carbohydrate recognition domain, exhibiting 97.6% amino acid identity in this region. We demonstrate that the 4D11 and Cwy-3 Abs, thought to be Ly-49G specific, also recognize Ly-49W. Rat RNK-16 cells transfected with Ly-49W mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49W can activate NK-mediated lysis. We further show that Ly-49W is allo-MHC specific: Ly-49W transfectants of RNK-16 only lysed Con A blasts expressing H-2k or H-2d haplotypes, and Ab-blocking experiments indicated that H-2Dk and Dd are ligands for Ly-49W. Ly-49W is the first activating Ly-49 receptor demonstrated to recognize an H-2k class I product. Ly-49G and Ly-49W represent a new pair of NK receptors with very similar ligand-binding domains, but opposite signaling functions.
Transactivation of C-Reactive Protein by IL-6 Requires Synergistic Interaction of CCAAT/Enhancer Binding Protein β (C/EBPβ) and Rel p50
Authors: Agrawal Alok , Cha-Molstad Hyunjoo , Samols David , Kushner Irving
Published: April 2014

We have previously found that overexpression of the Rel protein p50 stimulated C-reactive protein (CRP) expression in Hep 3B cells and that p50 could bind to a nonconsensus κB site overlapping the CCAAT/enhancer binding protein (C/EBP) binding site centered at position −53 on the CRP promoter. Accordingly, we employed EMSA to investigate possible cooperation between p50 and C/EBP proteins using an oligonucleotide probe (−63/−41) derived from the CRP promoter and containing both C/EBP and p50 binding sites. Abs to p50, but not to p65, decreased formation of C/EBPβ-containing complexes in nuclei of IL-6-treated cells, indicating that ternary complexes containing C/EBPβ and p50 are formed on the CRP promoter. Depletion of free Rel proteins by pretreatment of nuclear extracts with a κB consensus oligonucleotide markedly decreased formation of C/EBP complexes, indicating that Rel proteins are required for formation of such complexes. Overexpression of p50 in transient cotransfection studies using the proximal CRP promoter (−125/+9) linked to a luciferase reporter caused a 3-fold increase of luciferase activity, while C/EBPβ overexpression caused an 18-fold increase; simultaneous overexpression of both transcription factors increased luciferase activity ∼600-fold. Mutation of either the C/EBP binding site or the p50 binding site drastically reduced the effects of overexpressed transcription factors. Taken together, our findings indicate that binding of Rel p50 to the nonconsensus κB site enhances and stabilizes binding of C/EBPβ to the CRP promoter and that binding of both C/EBPβ and p50 to their overlapping cognate sites is required for induction of CRP expression by IL-6.
A Critical Role for CD2 in Both Thymic Selection Events and Mature T Cell Function
Authors: Sasada Tetsuro , Reinherz Ellis L.
Published: April 2014

To examine the function of CD2 in vivo, N15 TCR transgenic (tg) RAG-2−/− H-2b mice bearing a single TCR specific for the vesicular stomatitis virus octapeptide bound to the H-2Kb molecule were compared on a wild-type or CD2−/− background. In N15tg RAG-2−/− CD2−/− mice, thymic dysfunction is evident by 6 wk with a pre-TCR block in the CD4−CD8− double-negative thymocytes at the CD25+CD44− stage. Moreover, mature N15tg RAG-2−/− CD2−/− T cells are ∼100-fold less responsive to vesicular stomatitis virus octapeptide and unresponsive to weak peptide agonists, as judged by IFN-γ production. Repertoire analysis shows substantial differences in Vα usage between non-tg C57BL/6 (B6) and B6 CD2−/− mice. Collectively, these findings show that CD2 plays a role in pre-TCR function in double-negative thymocytes, TCR selection events during thymocyte development, and TCR-stimulated cytokine production in mature T cells.
Thrombospondin-1 Inhibits TCR-Mediated T Lymphocyte Early Activation
Authors: Li Zhuqing , He Liusheng , Wilson Katherine E. , Roberts David D.
Published: April 2014

Biological activities of the matrix glycoprotein thrombospondin-1 (TSP1) are cell type specific and depend on the relative expression or activation of several TSP1 receptors. Although engaging individual TSP1 receptors in T lymphocytes can elicit costimulating signals, in this study we show that intact TSP1 inhibits TCR-mediated T cell activation, assessed globally using cDNA microarrays. TSP1 signaling suppressed expression of several genes induced in Jurkat T cells, including the T cell activation markers CD69, early growth response gene-1 (Egr-1), and phosphatase of activated cells (PAC-1). TCR-stimulated and CD47-costimulated IL-2 secretion and cell surface CD69 expression were also inhibited by TSP1. The specific inhibitory effect of TSP1 was verified in freshly isolated human PBMCs. TSP1 inhibited TCR-mediated but not protein kinase C-mediated T cell activation. Using CD69 expression as a marker, we demonstrated that the inhibitory activity of TSP1 depended on two TSP1 receptors, CD47 and integrin-associated protein heparan sulfate proteoglycans. Signals from these receptors inhibited TCR signaling downstream of ZAP70, but upstream of NF-AT. Therefore, the expression of TSP1 induced during wound repair and in tumor stroma may limit T cell activation at these sites.
Identification of a CD28 Response Element in the CD40 Ligand Promoter
Authors: Parra Eduardo , Mustelin Tomas , Dohlsten Mikael , Mercola Dan
Published: April 2014

Ligation of the T cell coreceptor CD28 or CD2 by its cognate ligands B7-1 or LFA-3, respectively, greatly aids the Ag-induced up-regulation of several genes, including IL-2 and CD40 ligand (CD40L). Using luciferase reporter constructs under the control of the 1.2 kb of 5′ noncoding region of the human CD40L gene, we have found that stimulation through CD28 was required for a strong transcriptional activity of the CD40L promoter in response to TCR ligation, while the activity induced by CD2 was slightly lower than CD28. Deletion analysis demonstrated that the transcriptional elements mediating this effect were located within a 300-bp region upstream of the start site. Further dissection of this region and gel shift analyses demonstrated the presence of a CD28 response element in a region located between nucleotides −170 to −164 relative to the start site. Transcriptional studies with a CD40L enhancer-promoter carrying a mutation in this putative CD28 response element revealed that the activity was reduced by 80 and 70% after B7-1 and LFA-3 costimulation, respectively. The transcription factor complex bound to this site contained at least JunD, c-Fos, p50, p65, and c-Rel, but not c-Jun. Mutations introduced into the CD28RE also blocked the binding of this complex. These observations identify an important role for the CD28 signaling pathway in the regulation of CD40L promoter transcriptional activity.
Cloning of Human Preprotachykinin-I Promoter and the Role of Cyclic Adenosine 5′-Monophosphate Response Elements in Its Expression by IL-1 and Stem Cell Factor
Authors: Qian Jing , Yehia Ghassan , Molina Carlos A. , Fernandes Annemarie , Donnelly Robert J. , Anjaria Devashish J. , Gascon Pedro , Rameshwar Pranela
Published: April 2014

Preprotachykinin-I gene (PPT-I) encodes several peptides with organ-specific functions that link the neuroendocrine-immune-hemopoietic axis. We cloned upstream of the initiation site of human PPT-I promoter and identified consensus sequences for two cAMP response elements (CRE). PPT-I is induced by cytokines including those that signal through the cAMP pathway. Therefore, we studied the role of the two CRE in IL-1α and stem cell factor (SCF) stimulation of bone marrow stroma because both cytokines induce endogenous PPT-I in these cells and activate the cAMP pathway. Furthermore, bone marrow stroma expresses the transcription factors regulated by the cAMP pathways such as the repressor (ICERIIγ) and activator (CREMτ). Mutagenesis of the two CRE and/or cotransfection with vectors that express ICERIIγ or CREMτ indicated that the two CRE have major roles in PPT-I expression. The two CRE are also required for optimal promoter activity by SCF and IL-1α. A particular cytokine could concomitantly induce PPT-I and the high affinity G protein-coupled receptor for PPT-I peptides, NK-1R. We showed that SCF, a representative cytokine, induced PPT-I and NK-1R leading to autocrine and/or paracrine cell activation. Because NK-1R activates cAMP through the G protein, the results suggest that the presence of CRE sequences within PPT-I promoter could be important in the regulation of PPT-I expression by cytokines, irrespective of their ability to signal through cAMP. As PPT-I is implicated in hemopoietic regulation, immune responses, breast cancer, and other neural functions, these studies add to the basic biology of these processes and could provide targets for drug development.
The A′ and F′ Pockets of Human CD1b Are Both Required for Optimal Presentation of Lipid Antigens to T Cells
Authors: Niazi Kayvan R. , Chiu Melvin W. , Mendoza Richard M. , Degano Massimo , Khurana Sumit , Moody D. Branch , Melián Agustín , Wilson Ian A. , Kronenberg Mitchell , Porcelli Steven A. , Modlin Robert L.
Published: April 2014

CD1 proteins are unique in their ability to present lipid Ags to T cells. Human CD1b shares significant amino acid homology with mouse CD1d1, which contains an unusual putative Ag-binding groove formed by two large hydrophobic pockets, A′ and F′. We investigated the function of the amino acid residues that line the A′ and F′ pockets of CD1b by engineering 36 alanine-substitution mutants and analyzing their ability to present mycobacterial glycolipid Ags. Two lipid Ags presented by CD1b were studied, a naturally occurring glucose monomycolate (GMM) isolated from mycobacteria, which contains two long alkyl chains (C54-C62 and C22-C24) and synthetic GMM (sGMM), which includes two short alkyl chains (C18 and C14). We identified eight residues in both the A′ and F′ pockets that were involved in the presentation of both GMM and sGMM to T cells. Interestingly, four additional residues located in the distal portion of the A′ pocket were required for the optimal presentation of GMM, but not sGMM. Conversely, nine residues located between the center of the groove and the F′ pocket were necessary for the optimal presentation of sGMM, but not GMM. These data indicate that both the A′ and F′ pockets of human CD1b are required for the presentation of lipid Ags to T cells.
Engineered Antibodies with Increased Activity to Recruit Complement
Authors: Idusogie Esohe E. , Wong Pin Yee , Presta Leonard G. , Gazzano-Santoro Helene , Totpal Klara , Ultsch Mark , Mulkerrin Michael G.
Published: April 2014

This manuscript describes two sites in a human IgG1 that, when mutated individually or in combination, result in a dramatic increase in C1q binding and complement-dependent cytotoxicity activity. These two residues, K326 and E333, are located at the extreme ends of the C1q binding epicenter in the CH2 domain of a human IgG. A mutation to tryptophan at K326 debilitates Ab-dependent cell-mediated cytotoxicity activity. In addition, substitutions of the residues E333 with serine and of K326 with tryptophan in a human IgG2 confer biological activity in the complement-dependent cytotoxicity assay in which the wild-type IgG2 is inactive. This study reveals that the residues K326 and E333 play a significant role in the control of the biological activity of an IgG molecule and can rescue the activity of an inactive IgG isotype.
A Redundant Role of the CD3γ-Immunoreceptor Tyrosine-Based Activation Motif in Mature T Cell Function
Authors: Haks Mariëlle C. , Cordaro Tanina A. , van den Brakel Jeroen H. N. , Haanen John B. A. G. , de Vries Evert F. R. , Borst Jannie , Krimpenfort Paul , Kruisbeek Ada M.
Published: April 2014

At least four different CD3 polypeptide chains are contained within the mature TCR complex, each encompassing one (CD3γ, CD3δ, and CD3ε) or three (CD3ζ) immunoreceptor tyrosine-based activation motifs (ITAMs) within their cytoplasmic domains. Why so many ITAMs are required is unresolved: it has been speculated that the different ITAMs function in signal specification, but they may also serve in signal amplification. Because the CD3ζ chains do not contribute unique signaling functions to the TCR, and because the ITAMs of the CD3-γδε module alone can endow the TCR with normal signaling capacity, it thus becomes important to examine how the CD3γ-, δ-, and ε-ITAMs regulate TCR signaling. We here report on the role of the CD3γ chain and the CD3γ-ITAM in peripheral T cell activation and differentiation to effector function. All T cell responses were reduced or abrogated in T cells derived from CD3γ null-mutant mice, probably because of decreased expression levels of the mature TCR complex lacking CD3γ. Consistent with this explanation, T cell responses proceed undisturbed in the absence of a functional CD3γ-ITAM. Loss of integrity of the CD3γ-ITAM only slightly impaired the regulation of expression of activation markers, suggesting a quantitative contribution of the CD3γ-ITAM in this process. Nevertheless, the induction of an in vivo T cell response in influenza A virus-infected CD3γ-ITAM-deficient mice proceeds normally. Therefore, if ITAMs can function in signal specification, it is likely that either the CD3δ and/or the CD3ε chains endow the TCR with qualitatively unique signaling functions.
A Particular TCR β Variable Region Used by T Cells Infiltrating Kidney Transplants
Authors: Baron Christophe , Sachs David H. , LeGuern Christian
Published: April 2014

Immune tolerance to MHC class II identical renal grafts is achievable in miniature swine following a short immunosuppressive treatment. Like in clinical transplants, swine-accepted allografts are primarily infiltrated by CD8+ T cells, which are noncytotoxic to the renal tissue. However, the actual specificity and function of these intragraft-infiltrating lymphocytes remain poorly understood. To develop the molecular tools to study TCR-associated functions of graft-infiltrating cells in a preclinical transplantation model, we have determined the nucleotide sequence of 19 pig Vβ, 12 Jβ, and two Dβ. Sequence comparisons identified 17 different Vβ families and two Jβ clusters homologous to the human Jβ1 and Jβ2. The fact that the pig Jβ1 segments were always found joined to the Dβ1-like sequence in numerous rearranged TCR β cDNA suggests the existence of two d-J clusters in swine. These results also imply that the polymorphism of the porcine TCR β segments is similar to that found in human. Finally, we report the discovery of a new and functional Vβ subfamily named Vβ100, which exhibited similarity to the murine Vβ2 sequence but had no described Vβ homolog in humans. Pilot spectratyping studies on Vβ usage revealed a clonal dominance of Vβ100+ T cell subsets among infiltrating cells in two accepted grafts.
Formation of High-Affinity C5 Convertases of the Alternative Pathway of Complement
Authors: Rawal Nenoo , Pangburn Michael K.
Published: April 2014

Cleavage of C5 by C5 convertase is the last enzymatic step in the complement activation cascade leading to the formation of the cytolytic proteolytically activated form of C5 (C5b)-9 complex. In the present study, we examined the effect of the density of C3b (the proteolytically activated form of C3) on the function of the noncatalytic subunit of natural surface-bound forms of the enzyme. A comparison of the kinetic parameters of C5 convertases assembled on three surfaces (zymosan, rabbit erythrocytes, and sheep erythrocytes) were similar and revealed that the average Km decreased ∼28-fold (5.2–0.18 μM) when the density of C3b was increased from ∼18,000 to 400,000 C3b/cell. Very-high-affinity C5 convertases were generated when preformed C3 convertases were allowed to self amplify by giving them excess C3. These convertases exhibited Km from 0.016 to 0.074 μM, well below the normal plasma concentration of C5 in blood (0.37 μM). The results suggest that in serum convertases formed with monomeric C3b will be relatively inefficient in capturing C5 but will continue to cleave C3 opsonizing the cell surface for phagocytosis, whereas convertases formed with C3b-C3b complexes in areas of high C3b density will primarily cleave C5. The catalytic rate of these convertases approaches maximum velocity, thereby switching the enzyme from cleavage of C3 to cleavage of C5, and production of the cytolytic C5b-9 complex.
Elastase Controls the Binding of the Vitamin D-Binding Protein (Gc-Globulin) to Neutrophils: A Potential Role in the Regulation of C5a Co-Chemotactic Activity
Authors: DiMartino Stephen J. , Shah Anisha B. , Trujillo Glenda , Kew Richard R.
Published: April 2014

The vitamin D-binding protein (DBP) binds to the plasma membranes of numerous cell types and mediates a diverse array of cellular functions. DBP bound to the surface of leukocytes serves as a co-chemotactic factor for C5a, significantly enhancing the chemotactic activity of pM concentrations of C5a. This study investigated the regulation of DBP binding to neutrophils as a possible key step in the process of chemotaxis enhancement to C5a. Using radioiodinated DBP as a probe, neutrophils released 70% of previously bound DBP into the extracellular media during a 60-min incubation at 37°C. This was suppressed by serine protease inhibitors (PMSF, Pefabloc SC), but not by metallo- or thiol-protease inhibitors. DBP shed from neutrophils had no detectable alteration in its m.w., suggesting that a serine protease probably cleaves the DBP binding site, releasing DBP in an unaltered form. Cells treated with PMSF accumulate DBP vs time with over 90% of the protein localized to the plasma membrane. Purified neutrophil plasma membranes were used to screen a panel of protease inhibitors for their ability to suppress shedding of the DBP binding site. Only inhibitors to neutrophil elastase prevented the loss of membrane DBP-binding capacity. Moreover, treatment of intact neutrophils with elastase inhibitors prevented the generation of C5a co-chemotactic activity from DBP. These results indicate that steady state binding of DBP is essential for co-chemotactic activity, and further suggest that neutrophil elastase may play a critical role in the C5a co-chemotactic mechanism.
Expression of the Th1 Chemokine IFN-γ-Inducible Protein 10 in the Airway Alters Mucosal Allergic Sensitization in Mice
Authors: Wiley Ryan E. , Palmer Kay , Gajewska Beata U. , Stämpfli Martin R. , Alvarez David , Coyle Anthony J. , Gutierrez-Ramos José-Carlos , Jordana Manel
Published: April 2014

Although the preliminary characterization of chemokines and their receptors has been prolific, comparatively little is known about the role of chemokines in the evolution of immune responses. We speculate that the preferential recruitment of a particular immune cell population has implications for the short- and long-term features of an adaptive response. To test this hypothesis, we employed adenovirus-mediated gene transfer to express the Th1-affiliated, CXC chemokine IFN-γ-inducible protein (IP) 10 in the airways of mice undergoing a mucosal sensitization regimen known to result in a Th2-polarized allergic response. This resulted in a ∼60–75% inhibition of eosinophils in the bronchoalveolar lavage (BAL); these inflammatory changes were accompanied by enhanced IFN-γ, ablated IL-4, and, peculiarly, unaltered IL-5 and eotaxin levels in the BAL. The effect of IP-10 expression was shown to be dependent on IFN-γ, as there was no statistically significant reduction in BAL eosinophilia in IFN-γ knockout mice subjected to the IP-10 intervention. Flow cytometric analysis of mononuclear cells in the lung revealed a ∼60% reduction in the fraction of CD4+ cells expressing T1/ST2, a putative Th2 marker, and a parallel increase in the proportion expressing intracellular IFN-γ following IP-10 treatment. The effect of IP-10 expression at the time of initial Ag encounter is persistent, as mice rechallenged with OVA following the resolution of acute inflammation exhibited reduced eosinophilia and IL-4 in the BAL. Collectively, these data illustrate that local expression of the chemokine IP-10 can introduce Th1 phenomena to a Th2-predisposed context and subvert the development of a Th2 response.
Mast Cell Tissue Inhibitor of Metalloproteinase-1 Is Cleaved and Inactivated Extracellularly by α-Chymase
Authors: Frank Brendon T. , Rossall J. Caleb , Caughey George H. , Fang Kenneth C.
Published: April 2014

We previously reported that mast cell α-chymase cleaves and activates progelatinase B (progel B). Outside of cells, progel B is complexed with tissue inhibitor of metalloproteinase (TIMP)-1, which hinders zymogen activation and inhibits activity of mature forms. The current work demonstrates that dog BR mastocytoma cells, HMC-1 cells, and murine bone marrow-derived mast cells secrete TIMP-1 whose electrophoretic profile in supernatants suggests degranulation-dependent proteolysis. α-Chymase cleaves uncomplexed TIMP-1, reducing its ability to inhibit gel B, whereas tryptase has no effect. Sequencing of TIMP-1’s α-chymase-mediated cleavage products reveals hydrolysis at Phe12-Cys13 and Phe23-Val24 in loop 1 and Phe101-Val102 and Trp105-Asn106 in loop 3 of the NH2-terminal domain. TIMP-1 in a ternary complex with progel B and neutrophil gelatinase-associated lipocalin is also susceptible to α-chymase cleavage, yielding products like those resulting from processing of free TIMP-1. Thus, α-chymase cleaves free and gel B-bound TIMP-1. Incubation of the progel B-TIMP-1-neutrophil gelatinase-associated lipocalin complex with α-chymase increases gel B activity 2- to 5-fold, suggesting that α-chymase activates progel B whether it exists as free monomer or as a complex with TIMP-1. Furthermore, inhibition of α-chymase blocks degranulation-induced TIMP-1 processing (absent in α-chymase-deficient HMC-1 cells). Purified α-chymase processes TIMP-1 in BR supernatants, generating products like those induced by degranulation. In summary, these results suggest that controlled exocytosis of mast cell α-chymase activates progel B even in the presence of TIMP-1. This is the first identification of a protease that overcomes inhibition by bound TIMP-1 to activate progel B without involvement of other proteases.
Effect of Thymectomy on Human Peripheral Blood T Cell Pools in Myasthenia Gravis
Authors: Sempowski Gregory D. , Thomasch James R. , Gooding Maria E. , Hale Laura P. , Edwards Lloyd J. , Ciafaloni Emma , Sanders Donald B. , Massey Janice M. , Douek Daniel C. , Koup Richard A. , Haynes Barton F.
Published: April 2014

The human thymus is required for establishment of the T cell pool in fetal life, but postnatal thymectomy does not lead to immunodeficiency in humans. Because thymectomy in humans is performed for treatment of myasthenia gravis (MG), we have studied patients with MG for effects of thymectomy on peripheral blood (PB) naive (CD45RA+, CD62L+) and memory (CD45RO+) T cells. We have also determined the effect of thymectomy on levels of PB cells containing signal joint TCR δ excision circles (TRECs), a molecular marker of thymus emigrants that have divided few times after leaving the thymus. In 17 nonthymectomized and 26 thymectomized MG patients studied at varying times after thymectomy (1 day to 41 years), we found no significant mean difference in PB T cell TREC levels between ages 40 and 80 years. However, both thymectomized and nonthymectomized MG patients had lower PB T cell TREC levels than did age-matched normal subjects (p < 0.0001 for both). These data demonstrated that MG itself or treatment for MG decreased thymopoiesis independent of thymectomy. Next, to control for disease activity and treatment, we prospectively studied 10 MG patients before and from 27 to 517 days after thymectomy. We found that thymectomy decreased CD4 or CD8 T cell TREC concentrations most when thymopoiesis was active before thymectomy (six of six patients), but had little effect in patients when thymopoiesis was minimal (four of four patients). In contrast, there was no significant effect of thymectomy on absolute numbers of naive PB T cells. Thus, in MG, removal of a thymus with active thymopoiesis resulted in a significant fall in PB TREC+ T cells postthymectomy.
Human B Cell Growth and Differentiation in the Spleen of Immunodeficient Mice
Authors: Depraetere Stany , Verhoye Lieven , Leclercq Georges , Leroux-Roels Geert
Published: April 2014

Human mAbs (HumAbs) have therapeutic potential against infectious diseases and cancer. Heretofore, their production has been hampered by ethical constraints preventing the isolation of Ag-specific activated B cells by in vivo immunization. Alternatively, severe combined immune deficient (SCID) mice, transplanted i.p. with human (Hu)-PBLs, allow the in vivo stimulation of human Ab responses without the usual constraints. Unfortunately, human B cells only represent a minor fraction of the surviving graft, they are scattered all over the animal body, and thus are hard to isolate for subsequent immortalization procedures. To prevent this dispersion and to provide the human B cells with a niche for expansion and maturation, SCID mice were engrafted with Hu-PBL directly into the spleen. Simultaneously endogenous murine NK cell activity was depleted by treatment with an anti-mouse IL-2 receptor β-chain Ab. During engraftment, human B lymphocytes became activated, divided intensely, and differentiated into plasmacytoid cells. In vivo exposure to a recall Ag after cell transfer induced expansion of Ag-specific B cell clones. One week after inoculation, human B cells were abundant in the spleen and could easily be recovered for fusion with a heteromyeloma line. This resulted in the formation of stable hybridoma cell lines that secreted Ag-specific HumAbs. Thus transplantation of human lymphoid cells in the spleens of immune deficient mice represents a model for the study of human T cell-dependent B cell activation and proves to be an excellent tool for the successful production of HumAbs.
Thioredoxin Acts as a B Cell Growth Factor in Channel Catfish
Authors: Khayat Morad , Stuge Tor B. , Wilson Melanie , Bengtén Eva , Miller Norman W. , Clem L. William
Published: April 2014

To identify differentially expressed genes from channel catfish macrophages, a cDNA library from LPS-stimulated catfish macrophages was screened by subtractive hybridization. This screening yielded a 552-bp cDNA coding for catfish thioredoxin (CF-TRX). The deduced amino acid sequence revealed that CF-TRX contains 107 amino acids and is 59% homologous to human adult T cell leukemia-derived factor/TRX, originally described as an IL-2Rα-inducing factor. Northern blot analyses showed that CF-TRX is expressed in catfish T and macrophage cell lines, but weakly in B cell lines. Similar results were also observed in Western blot analyses using a mAb specific for recombinant CF-TRX (rTRX). The use of rTRX in functional studies demonstrated that rTRX induces in vitro proliferative responses of catfish PBL that were synergistically enhanced by the addition of culture supernatants from catfish T cell lines. In addition, cell separation studies and flow cytometric analyses revealed that the cells proliferating in rTRX-stimulated cultures were mostly B cells. These results suggest that CF-TRX may have an important role in the activation and proliferation of channel catfish B cells.
Induction of Polyclonal Prostate Cancer-Specific CTL Using Dendritic Cells Transfected with Amplified Tumor RNA
Authors: Heiser Axel , Maurice Margaret A. , Yancey Donna R. , Wu Ning Z. , Dahm Philipp , Pruitt Scott K. , Boczkowski David , Nair Smita K. , Ballo Michael S. , Gilboa Eli , Vieweg Johannes
Published: April 2014

Polyvalent cancer vaccines targeting the entire antigenic spectrum on tumor cells may represent a superior therapeutic strategy for cancer patients than vaccines solely directed against single Ags. In this study, we show that autologous dendritic cells (DC) transfected with RNA amplified from microdissected tumor cells are capable of stimulating CTL against a broad set of unidentified and critical prostate-specific Ags. Although the polyclonal CTL responses generated with amplified tumor RNA-transfected DC encompassed as a subcomponent a response against prostate-specific Ag (PSA) as well as against telomerase reverse transcriptase, the tumor-specific CTL were consistently more effective than PSA or telomerase reverse transcriptase CTL to lyse tumor targets, suggesting the superiority of the polyclonal response. Although tumor RNA-transfected DC stimulated CTL, which recognized not only tumor but also self-Ags expressed by benign prostate tissue, these cross-reactive CTL were exclusively specific for the PSA, indicating an immunodominant role of PSA in the prostate cancer-specific immune response. Our data suggest that tumor RNA-transfected DC may represent a broadly applicable, potentially clinically effective vaccine strategy for prostate cancer patients, which is not limited by tumor tissue availability for Ag preparation and may minimize the risk of clonal tumor escape.