bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of IL-5 for Mature B-1 Cells in Homeostatic Proliferation, Cell Survival, and Ig Production
Authors: Moon Byoung-gon , Takaki Satoshi , Miyake Kensuke , Takatsu Kiyoshi
Published: April 2014

B-1 cells, distinguishable from conventional B-2 cells by their cell surface marker, anatomical location, and self-replenishing activity, play an important role in innate immune responses. B-1 cells constitutively express the IL-5R α-chain (IL-5Rα) and give rise to Ab-producing cells in response to various stimuli, including IL-5 and LPS. Here we report that the IL-5/IL-5R system plays an important role in maintaining the number and the cell size as well as the functions of mature B-1 cells. The administration of anti-IL-5 mAb into wild-type mice, T cell-depleted mice, or mast cell-depleted mice resulted in reduction in the total number and cell size of B-1 cells to an extent similar to that of IL-5Rα-deficient (IL-5Rα−/−) mice. Cell transfer experiments have demonstrated that B-1 cell survival in wild-type mice and homeostatic proliferation in recombination-activating gene 2-deficient mice are impaired in the absence of IL-5Rα. IL-5 stimulation of wild-type B-1 cells, but not IL-5Rα−/− B-1 cells, enhances CD40 expression and augments IgM and IgG production after stimulation with anti-CD40 mAb. Enhanced IgA production in feces induced by the oral administration of LPS was not observed in IL-5Rα−/− mice. Our results illuminate the role of IL-5 in the homeostatic proliferation and survival of mature B-1 cells and in IgA production in the mucosal tissues.
The Ability of Two Listeria monocytogenes Vaccines Targeting Human Papillomavirus-16 E7 to Induce an Antitumor Response Correlates with Myeloid Dendritic Cell Function
Authors: Peng Xiaohui , Hussain S. Farzana , Paterson Yvonne
Published: April 2014

Previous work from our laboratory has shown that Lm-LLO-E7 induces complete regression of ∼75% of established TC-1 tumors, whereas Lm-E7 only slows the growth of such tumors. In this study, we examine the effects of Lm-LLO-E7 vs Lm-E7 on APCs. We hypothesize that the difference in antitumor efficacy of the two vaccines is due to the ability of each of these vectors to render immature dendritic cells (DCs) effective APCs in terms of MHC class II or costimulatory molecule expression. We also examine the ability of these vectors to stimulate cytokine production by DCs. Both vectors induced IL-12 and TNF-α, but only Lm-LLO-E7 induced IL-2 production by DCs. Lm-LLO-E7 also induced significantly higher levels of MHC class II molecules, CD40, and B7 costimulatory molecules (CD86, B7-H1, and B7-DC) on DCs than Lm-E7. Interestingly, a shift of CD11c+ cells from CD86low to CD86high is observed post-Lm-LLO-E7 infection. A similar shift is also observed for B7-H1 and B7-DC molecules. Moreover, Lm-LLO-E7, but not Lm-E7-pulsed DCs, stimulate naive T cell proliferation. These results indicate that Lm-LLO-E7 is more effective than Lm-E7 at inducing DC maturation. This effect is independent of the E7 Ag, because Lm-LLO-NP, and a mixture of Lm-LLO-NP and Lm-E7 induce the same changes in DC phenotype as Lm-LLO-E7. Taken together, the changes in DC expression correlate well with the differences in antitumor efficacy between these two vaccines.
T Cells Associated with Tumor Regression Recognize Frameshifted Products of the CDKN2A Tumor Suppressor Gene Locus and a Mutated HLA Class I Gene Product
Authors: Huang Jianping , El-Gamil Mona , Dudley Mark E. , Li Yong F. , Rosenberg Steven A. , Robbins Paul F.
Published: April 2014

The dramatic tumor regression observed following adoptive T cell transfer in some patients has led to attempts to identify novel Ags to understand the nature of these responses. Nearly complete regression of multiple metastatic melanoma lesions was observed in patient 1913 following adoptive transfer of autologous tumor-infiltrating lymphocytes. The autologous 1913 melanoma cell line expressed a mutated HLA-A11 class I gene product that was recognized by the bulk tumor-infiltrating lymphocytes as well as a dominant T cell clone derived from this line. A second dominant T cell clone, T1D1, did not recognize the mutated HLA-A11 product, but recognized an allogeneic melanoma cell line that shared expression of HLA-A11 with the parental tumor cell line. Screening of an autologous melanoma cDNA library with clone T1D1 T cells in a cell line expressing the mutated HLA-A11 gene product resulted in the isolation of a p14ARF transcript containing a 2-bp deletion in exon 2. The T cell epitope recognized by T1D1, which was encoded within the frameshifted region of the deleted p14ARF transcript, was also generated from frameshifted p14ARF or p16INK4a transcripts that were isolated from two additional melanoma cell lines. The results of monitoring studies indicated that T cell clones reactive with the mutated HLA-A11 gene product and the mutated p14ARF product were highly represented in the peripheral blood of patient 1913 1 wk following adoptive transfer, indicating that they may have played a role in the nearly complete tumor regression that was observed following this treatment.
Toll-Like Receptor Ligands Directly Promote Activated CD4+ T Cell Survival
Authors: Gelman Andrew E. , Zhang Jidong , Choi Yongwon , Turka Laurence A.
Published: April 2014

Toll-like receptor (TLR) engagement by pathogen-associated molecular patterns (PAMPs) is an important mechanism for optimal cellular immune responses. APC TLR engagement indirectly enhances activated CD4+ T cell proliferation, differentiation, and survival by promoting the up-regulation of costimulatory molecules and the secretion of proinflammatory cytokines. However, TLRs are also expressed on CD4+ T cells, suggesting that PAMPs may also act directly on activated CD4+ T cells to mediate functional responses. In this study, we show that activated mouse CD4+ T cells express TLR-3 and TLR-9 but not TLR-2 and TLR-4. Treatment of highly purified activated CD4+ T cells with the dsRNA synthetic analog poly(I:C) and CpG oligodeoxynucleotides (CpG DNA), respective ligands for TLR-3 and TLR-9, directly enhanced their survival without augmenting proliferation. In contrast, peptidoglycan and LPS, respective ligands for TLR-2 and TLR-4 had no effect. Enhanced survival mediated by either poly(I:C) or CpG DNA required NF-κB activation and was associated with Bcl-xL up-regulation. However, only CpG DNA, but not poly(I:C)-mediated effects on activated CD4+ T cells required the TLR/IL-1R domain containing adaptor molecule myeloid differentiation factor 88. Collectively, our results demonstrate that PAMPs can directly promote activated CD4+ T cell survival, suggesting that TLRs on T cells can directly modulate adaptive immune responses.
The Tumoricidal Activity of Memory CD8+ T Cells Is Hampered by Persistent Systemic Antigen, but Full Functional Capacity Is Regained in an Antigen-Free Environment
Authors: den Boer Annemieke Th. , van Mierlo Geertje J. D. , Fransen Marieke F. , Melief Cornelis J. M. , Offringa Rienk , Toes René E. M.
Published: April 2014

Naive T cells can be tolerized in the periphery by diverse mechanisms. However, the extent to which memory T cells are susceptible to tolerance induction is less well defined. Vaccination of mice with a minimal CTL epitope derived from human adenovirus type 5 E1A in IFA s.c. readily tolerizes naive as well as recently activated CD8+ T cells due to the overwhelming systemic and persistent presence of the peptide. We have now studied the effect of this peptide on established memory cells, which were induced at least 50 days before by virus vaccination. Memory cells did not undergo peripheral deletion and kept their ability to produce IFN-γ as well as their cytolytic activity in response to Ag directly ex vivo. However, memory CTL responses in virus vaccinated mice injected with peptide ceased to control tumor outgrowth. Interestingly, functional capacities were regained when T cells were transferred to an Ag-free environment in vivo as determined by their ability to reject an otherwise lethal tumor challenge. Together, these findings indicate that memory CTL responses can be functionally incapacitated, but are not, in contrast to naive or recently activated T cells, irreversibly tolerized by persistent systemic Ag, as memory T cells quickly regain effector function upon disappearance of the Ag.
IL-9-Induced Expansion of B-1b Cells Restores Numbers but Not Function of B-1 Lymphocytes in xid Mice
Authors: Knoops Laurent , Louahed Jamila , Renauld Jean-Christophe
Published: April 2014

Mice expressing the X-linked immunodeficiency (xid) mutation lack functional Bruton’s tyrosine kinase and were shown to be specifically deficient in peritoneal B-1 lymphocytes. We have previously shown that IL-9, a cytokine produced by TH2 lymphocytes, promotes B-1 cell expansion in vivo. To determine whether IL-9 overexpression might compensate the xid mutation for B-1 lymphocyte development, we crossed xid mice with IL-9-transgenic mice. In this model, IL-9 restored normal numbers of mature peritoneal B-1 cells that all belonged to the CD5− B-1b subset. Despite this normal B-1 lymphocyte number, IL-9 failed to restore classical functions of B-1 cells, namely, the production of natural IgM Abs, the T15 Id Ab response to phosphorylcholine immunization, and the antipolysaccharide humoral response against Streptococcus pneumoniae. By using bromelain-treated RBC, we showed that the antigenic repertoire of these IL-9-induced B-1b lymphocytes was different from the repertoire of classical CD5+ B-1a cells, indicating that the lack of B-1 function by B-1b cells is associated with distinct Ag specificities. Taken together, our data show that B-1b cell development can restore the peritoneal B-1 population in xid mice but that these B-1b cells are functionally distinct from CD5+ B-1a lymphocytes.
CD1d-Independent NKT Cells in β2-Microglobulin-Deficient Mice Have Hybrid Phenotype and Function of NK and T Cells
Authors: Maeda Motoi , Shadeo Ashleen , MacFadyen Anna M. , Takei Fumio
Published: April 2014

Unlike CD1d-restricted NK1.1+TCRαβ+ (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in β2-microglobulin (β2m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in β2m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-indpendent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed β2m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by Db killed RMA-S, but not RMA, cells. They also killed RMA (H-2b) cells that were preincubated with the HY peptide. NKT cells from β2m-deficient mice, upon CD3 cross-linking, secreted IFN-γ and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.
Human CD4+CD25+ Regulatory T Cells Share Equally Complex and Comparable Repertoires with CD4+CD25− Counterparts
Authors: Kasow Kimberly A. , Chen Xiaohua , Knowles James , Wichlan David , Handgretinger Rupert , Riberdy Janice M.
Published: April 2014

CD4+CD25+ T cells are critical mediators of peripheral immune tolerance. However, many developmental and functional characteristics of these cells are unknown, and knowledge of human regulatory T cells is particularly limited. To better understand how human CD4+CD25+ T cells develop and function, we examined the diversity of CD4+CD25+ and CD4+CD25− T cell repertoires in both thymus and peripheral blood. Levels of T receptor excision circles (TREC) were comparable in purified CD4+CD25+ and CD4+CD25− thymic populations, but were significantly higher than those in samples derived from peripheral blood, consistent with murine studies demonstrating thymic development of CD4+CD25+ regulatory T cells. Surprisingly, CD4+CD25− T cells isolated from peripheral blood had greater TREC quantities than their CD4+CD25+ counterparts, supporting the possibility of extrathymic expansion as well. CD4+CD25+ and CD4+CD25− T cells from a given individual showed overlapping profiles with respect to diversity by Vβ staining and spectratyping. Interestingly, CD4+CD25+ T cells have lower quantities of CD3 than CD4+CD25− T cells. Collectively, these data suggest that human CD4+CD25+ T cells recognize a similar array of Ags as CD4+CD25− T cells. However, reduced levels of TCR on regulatory T cells suggest different requirements for activation and may contribute to how the immune system regulates whether a particular response is suppressed or augmented.
Transcriptional Activation of the SH2D2A Gene Is Dependent on a Cyclic Adenosine 5′-Monophosphate-Responsive Element in the Proximal SH2D2A Promoter
Authors: Dai Ke-Zheng , Johansen Finn-Eirik , Kolltveit Kristin Melkevik , Aasheim Hans-Christian , Dembic Zlatko , Vartdal Frode , Spurkland Anne
Published: April 2014

The SH2D2A gene, encoding the T cell-specific adapter protein (TSAd), is rapidly induced in activated T cells. In this study we investigate the regulation of the SH2D2A gene in Jurkat T cells and in primary T cells. Reporter gene assays demonstrated that the proximal 1-kb SH2D2A promoter was constitutively active in Jurkat TAg T cells and, to a lesser extent, in K562 myeloid cells, Reh B cells, and 293T fibroblast cells. The minimal SH2D2A promoter was located between position −236 and −93 bp from the first coding ATG, and transcriptional activity in primary T cells depended on a cAMP response element (CRE) centered around position −117. Nuclear extracts from Jurkat TAg cells and activated primary T cells contained binding activity to this CRE, as observed in an EMSA. Consistent with this observation, we found that a cAMP analog was a very potent inducer of SH2D2A mRNA expression in primary T cells as measured by real-time RT-PCR. Furthermore, activation of SH2D2A expression by CD3 stimulation required cAMP-dependent protein kinase activity. Thus, transcriptional regulation of the SH2D2A gene in activated T cells is critically dependent on a CRE in the proximal promoter region.
Lipopolysaccharide-Binding Protein Critically Regulates Lipopolysaccharide-Induced IFN-β Signaling Pathway in Human Monocytes
Authors: Kato Atsushi , Ogasawara Takahisa , Homma Toshiki , Saito Hirohisa , Matsumoto Kenji
Published: April 2014

LPS binding to Toll-like receptor 4 induces a large number of genes through activation of NF-κB and IFN-regulatory factor-3 (IRF-3). However, no previous reports have tested the role of serum proteins in LPS-induced gene expression profiles. To investigate how serum proteins affect LPS-induced signaling, we investigated LPS-inducible genes in PBMC using an oligonucleotide probe-array system. Approximately 120 genes up-regulated by LPS were hierarchically divided into two clusters. Induction of one cluster, containing only IFN-inducible genes, was serum dependent. Real-time PCR analysis confirmed that IFN-inducible genes were induced only in the presence of serum, whereas inflammatory genes were induced both in the presence and absence of serum. Further analysis demonstrated that addition of LPS-binding protein (LBP), but not of soluble CD14 to the serum-free medium enabled the induction of IFN-inducible genes and IFN-β itself by LPS in human monocytes. The mRNAs for IFN-β and IFN-inducible genes were induced by LPS only in the presence of serum from LBP+/+ mice, and not in the presence of serum from LBP−/− mice. Blocking experiments also confirmed the involvement of LBP in this phenomenon. Immunoblotting analysis showed that phosphorylation of c-Jun N-terminal kinase, p38, IRF-3, tyrosine kinase 2, and STAT1 by LPS, but not of NF-κB and extracellular signal-regulated kinase was abrogated in the absence of LBP. This critical role for LBP implies the presence of possible mechanisms linking LBP to the intracellular signaling between Toll-like receptor 4 and IRF-3, leading to the induction of IFN-β by LPS.
Lysine-Dependent Multipoint Binding of theBorrelia burgdorferiVirulence Factor Outer Surface Protein E to the C Terminus of Factor H
Authors: Alitalo Antti , Meri Taru , Chen Tong , Lankinen Hilkka , Cheng Zhu-Zhu , Jokiranta T. Sakari , Seppälä Ilkka J. T. , Lahdenne Pekka , Hefty P. Scott , Akins Darrin R. , Meri Seppo
Published: April 2014

Serum resistance, an important virulence determinant of Borrelia burgdorferi sensu lato strains belonging to the Borrelia afzelii and B. burgdorferi sensu stricto genotypes, is related to binding of the complement inhibitor factor H to the spirochete surface protein outer surface protein E (OspE) and its homologues. In this study, we show that the C-terminal short consensus repeats 18–20 of both human and mouse factor H bind to OspE. Analogously, factor H-related protein 1, a distinct plasma protein with three short consensus repeat domains homologous to those in factor H, bound to OspE. Deleting 15-aa residues (region V) from the C terminus of the OspE paralog P21 (a 20.7-kDa OspE-paralogous surface lipoprotein in the B. burgdorferi sensu stricto 297 strain) abolished factor H binding. However, C-terminal peptides from OspE, P21, or OspEF-related protein P alone and the C-terminal deletion mutants of P21 inhibited factor H binding to OspE only partially when compared with full-length P21 or its N-terminal mutant. Alanine substitution of amino acids in peptides from the key binding regions of the OspE family indicated that several lysine residues are required for factor H binding. Thus, the borrelial OspE family proteins bind the C inhibitor factor H via multiple sites in a lysine-dependent manner. The C-terminal site V (Ala151-Lys166) is necessary, but not sufficient, for factor H binding in both rodents and humans. Identification of the necessary binding sites forms a basis for the development of vaccines that block the factor H-OspE interaction and thereby promote the killing of Borreliae.
Filaria-Induced Immune Evasion: Suppression by the Infective Stage of Brugia malayi at the Earliest Host-Parasite Interface
Authors: Semnani Roshanak Tolouei , Law Melissa , Kubofcik Joseph , Nutman Thomas B.
Published: April 2014

To assess the physiologic interactions between the infective stage of Brugia malayi—one of the extracellular parasites responsible for lymphatic filariasis in humans—and the APC with which they come in contact during their development and routes of travel, we have investigated the interaction between the infective stage (L3) of B. malayi and human Langerhans cells (LC) in the skin. Our data indicate that live L3 result in increased migration of LC from the epidermis without affecting the viability of these cells and up-regulation of the IL-18 cytokine involved in LC migration. Live L3 also result in down-regulation of MHC class I and II on the LC cell surface. Additionally, microarray data indicate that live L3 significantly down-regulated expression of IL-8 as well as of multiple genes involved in Ag presentation, reducing the capacity of LC to induce CD4+ T cells in allogeneic MLR, and thus resulting in a decreased ability of LC to promote CD4+ T cell proliferation and production of IFN-γ and IL-10. These data suggest that L3 exert a down-regulatory response in epidermal LC that leads to a diminished capacity of these cells to activate CD4+ T cells.
Immunogenicity of Recombinant Adenovirus Serotype 35 Vaccine in the Presence of Pre-Existing Anti-Ad5 Immunity
Authors: Barouch Dan H. , Pau Maria G. , Custers Jerome H. H. V. , Koudstaal Wouter , Kostense Stefan , Havenga Menzo J. E. , Truitt Diana M. , Sumida Shawn M. , Kishko Michael G. , Arthur Janelle C. , Korioth-Schmitz Birgit , Newberg Michael H. , Gorgone Darci A. , Lifton Michelle A. , Panicali Dennis L. , Nabel Gary J. , Letvin Norman L. , Goudsmit Jaap
Published: April 2014

The high prevalence of pre-existing immunity to adenovirus serotype 5 (Ad5) in human populations may substantially limit the immunogenicity and clinical utility of recombinant Ad5 vector-based vaccines for HIV-1 and other pathogens. A potential solution to this problem is to use vaccine vectors derived from adenovirus (Ad) serotypes that are rare in humans, such as Ad35. However, cross-reactive immune responses between heterologous Ad serotypes have been described and could prove a major limitation of this strategy. In particular, the extent of immunologic cross-reactivity between Ad5 and Ad35 has not previously been determined. In this study we investigate the impact of pre-existing anti-Ad5 immunity on the immunogenicity of candidate rAd5 and rAd35 vaccines expressing SIV Gag in mice. Anti-Ad5 immunity at levels typically found in humans dramatically blunted the immunogenicity of rAd5-Gag. In contrast, even high levels of anti-Ad5 immunity did not substantially suppress Gag-specific cellular immune responses elicited by rAd35-Gag. Low levels of cross-reactive Ad5/Ad35-specific CD4+ T lymphocyte responses were observed, but were insufficient to suppress vaccine immunogenicity. These data demonstrate the potential utility of Ad35 as a candidate vaccine vector that is minimally suppressed by anti-Ad5 immunity. Moreover, these studies suggest that using Ad vectors derived from immunologically distinct serotypes may be an effective and general strategy to overcome the suppressive effects of pre-existing anti-Ad immunity.
Dual Role of TLR2 and Myeloid Differentiation Factor 88 in a Mouse Model of Invasive Group B Streptococcal Disease
Authors: Mancuso Giuseppe , Midiri Angelina , Beninati Concetta , Biondo Carmelo , Galbo Roberta , Akira Shizuo , Henneke Philipp , Golenbock Douglas , Teti Giuseppe
Published: April 2014

Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF-α and IL-6. Administration of anti-TNF-α Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF-α as the molecular mediator of bacterial clearance and septic shock.
Transient and Selective NF-κB p65 Serine 536 Phosphorylation Induced by T Cell Costimulation Is Mediated by IκB Kinase β and Controls the Kinetics of p65 Nuclear Import
Authors: Mattioli Ivan , Sebald Andrea , Bucher Cyril , Charles Roch-Philippe , Nakano Hiroyasu , Doi Takahiro , Kracht Michael , Schmitz M. Lienhard
Published: April 2014

Full transcriptional activity of the nuclear, DNA-bound form of NF-κB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-κB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IκBα and IκB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Cθ, and NF-κB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKβ, but not by IKKα. Phosphorylation occurs within the cytoplasmic and intact NF-κB/IκBα complex and requires prior phosphorylation of IκBα at serines 32 and 36. Reconstitution of p65−/− cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IκBα localization and the kinetics of p65 nuclear import.
Studies of Congenic Lines in the Brown Norway Rat Model of Th2-Mediated Immunopathological Disorders Show That the Aurothiopropanol Sulfonate-Induced Immunological Disorder (Aiid3) Locus on Chromosome 9 Plays a Major Role Compared toAiid2on Chromosome 10
Authors: Mas Magali , Cavaillès Pierre , Colacios Céline , Subra Jean-François , Lagrange Dominique , Calise Maryline , Christen Marie-Odile , Druet Philippe , Pelletier Lucette , Gauguier Dominique , Fournié Gilbert J.
Published: April 2014

Brown Norway (BN) rats treated with aurothiopropanol-sulfonate (Atps) constitute a model of Th2-mediated immunological disorders associated with elevated IgE responses and renal IgG deposits. Using F2 offspring between Atps-susceptible BN and Atps-resistant Lewis rats, we had previously mapped three quantitative trait loci on chromosomes 9, 10, and 20 for which BN alleles increased susceptibility to Atps-induced immunological disorders (Aiid). In this study we have used congenic lines for the latter two quantitative trait loci, formerly called Atps2 and Atps3 and now named Aiid2 (chromosome 10) and Aiid3 (chromosome 9), for fine mapping and characterization of their impact on Atps-triggered reactions. In Aiid2 congenic lines, the gene(s) controlling part of the IgE response to Atps was mapped to an ∼7-cM region, which includes the IL-4 cytokine gene cluster. Two congenic lines in which the introgressed segments shared only a portion of this 7-cM region, showed an intermediate IgE response, indicating the involvement of several genes within this region. Results from BN rats congenic for the Lewis Aiid3 locus, which we mapped to a 1.2-cM interval, showed a stronger effect of this region. In this congenic line, the Atps-triggered IgE response was 10-fold lower than in the BN parental strain, and glomerular IgG deposits were either absent or dramatically reduced. Further genetic and functional dissections of these loci should provide insights into pathways that lead to Th2-adverse reactions.
The Transmembrane CXC-Chemokine Ligand 16 Is Induced by IFN-γ and TNF-α and Shed by the Activity of the Disintegrin-Like Metalloproteinase ADAM10
Authors: Abel Soeren , Hundhausen Christian , Mentlein Rolf , Schulte Alexander , Berkhout Theo A. , Broadway Neil , Hartmann Dieter , Sedlacek Radek , Dietrich Sebastian , Muetze Barbara , Schuster Bjoern , Kallen Karl-Josef , Saftig Paul , Rose-John Stefan , Ludwig Andreas
Published: April 2014

The novel CXC-chemokine ligand 16 (CXCL16) functions as transmembrane adhesion molecule on the surface of APCs and as a soluble chemoattractant for activated T cells. In this study, we elucidate the mechanism responsible for the conversion of the transmembrane molecule into a soluble chemokine and provide evidence for the expression and shedding of CXCL16 by fibroblasts and vascular cells. By transfection of human and murine CXCL16 in different cell lines, we show that soluble CXCL16 is constitutively generated by proteolytic cleavage of transmembrane CXCL16 resulting in reduced surface expression of the transmembrane molecule. Inhibition experiments with selective hydroxamate inhibitors against the disintegrin-like metalloproteinases a disintegrin and metalloproteinase domain (ADAM)10 and ADAM17 suggest that ADAM10, but not ADAM17, is involved in constitutive CXCL16 cleavage. In addition, the constitutive cleavage of transfected human CXCL16 was markedly reduced in embryonic fibroblasts generated from ADAM10-deficient mice. By induction of murine CXCL16 in ADAM10-deficient fibroblasts with IFN-γ and TNF-α, we show that endogenous ADAM10 is indeed involved in the release of endogenous CXCL16. Finally, the shedding of endogenous CXCL16 could be reconstituted by retransfection of ADAM10-deficient cells with ADAM10. Analyzing the expression and release of CXCXL16 by cultured vascular cells, we found that IFN-γ and TNF-α synergize to induce CXCL16 mRNA. The constitutive shedding of CXCL16 from the endothelial cell surface is blocked by inhibitors of ADAM10 and is independent of additional inhibition of ADAM17. Hence, during inflammation in the vasculature, ADAM10 may act as a CXCL16 sheddase and thereby finely control the expression and function of CXCL16 in the inflamed tissue.
6-Phosphogluconate Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Form a Supramolecular Complex in Human Neutrophils That Undergoes Retrograde Trafficking during Pregnancy
Authors: Kindzelskii Andrei L. , Ueki Tatsuya , Michibata Hitoshi , Chaiworapongsa Tinnakorn , Romero Roberto , Petty Howard R.
Published: April 2014

Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite (ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubule-organizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme’s intracellular distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that 6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing ROM and NO production in comparison with fully activated neutrophils from nonpregnant women.
Inactivation of C5a Anaphylatoxin by a Peptide That Is Complementary to a Region of C5a
Authors: Fujita Emiko , Farkas Imre , Campbell William , Baranyi Lajos , Okada Hidechika , Okada Noriko
Published: April 2014

PL37 (RAARISLGPRCIKAFTE) is an antisense homology box peptide composed of aa 37–53 of C5a-anaphylatoxin and is considered to be the region essential for C5a function. Using a computer program, we designed the complementary peptides ASGAPAPGPAGPLRPMF (Pep-A) and ASTAPARAGLPRLPKFF (Pep-B). Pep-A bound to PL37 and to C5a with very slow dissociation as determined by analysis using surface plasmon resonance, whereas Pep-B failed to bind at all. C5a was inactivated by concentrations of 7 nM or more of Pep-A, and this concentration of Pep-A inhibited induction of intracellular Ca2+ influx in neutrophils. Patch clamp electrophysiology experiments also showed the effectiveness of Pep-A in C5aR-expressing neuroblastoma cells. Furthermore, Pep-A administration prevented rats from C5a-mediated rapid lethal shock induced by an Ab to a membrane inhibitor of complement after LPS sensitization.
The Functional Profile of Primary Human Antiviral CD8+ T Cell Effector Activity Is Dictated by Cognate Peptide Concentration
Authors: Betts Michael R. , Price David A. , Brenchley Jason M. , Loré Karin , Guenaga F. Javier , Smed-Sorensen Anna , Ambrozak David R. , Migueles Stephen A. , Connors Mark , Roederer Mario , Douek Daniel C. , Koup Richard A.
Published: April 2014

Antiviral CD8+ T cells can elaborate at least two effector functions, cytokine production and cytotoxicity. Which effector function is elaborated can determine whether the CD8+ T cell response is primarily inflammatory (cytokine producing) or antiviral (cytotoxic). In this study we demonstrate that cytotoxicity can be triggered at peptide concentrations 10- to 100-fold less than those required for cytokine production in primary HIV- and CMV-specific human CD8+ T cells. Cytolytic granule exocytosis occurs at peptide concentrations insufficient to cause substantial TCR down-regulation, providing a mechanism by which a CD8+ T cell could engage and lyse multiple target cells. TCR sequence analysis of virus-specific cells shows that individual T cell clones can degranulate or degranulate and produce cytokine depending on the Ag concentration, indicating that response heterogeneity exists within individual CD8+ T cell clonotypes. Thus, antiviral CD8+ T cell effector function is determined primarily by Ag concentration and is not an inherent characteristic of a virus-specific CD8+ T cell clonotype or the virus to which the response is generated. The inherent ability of viruses to induce high or low Ag states may be the primary determinant of the cytokine vs cytolytic nature of the virus-specific CD8+ T cell response.
CD4+CD25bright Regulatory T Cells Actively Regulate Inflammation in the Joints of Patients with the Remitting Form of Juvenile Idiopathic Arthritis
Authors: de Kleer Ismé M. , Wedderburn Lucy R. , Taams Leonie S. , Patel Alka , Varsani Hemlata , Klein Mark , de Jager Wilco , Pugayung Gisela , Giannoni Francesca , Rijkers Ger , Albani Salvatore , Kuis Wietse , Prakken Berent
Published: April 2014

This study investigates the role of CD4+CD25+ regulatory T cells during the clinical course of juvenile idiopathic arthritis (JIA). Persistent oligoarticular JIA (pers-OA JIA) is a subtype of JIA with a relatively benign, self-remitting course while extended oligoarticular JIA (ext-OA JIA) is a subtype with a much less favorable prognosis. Our data show that patients with pers-OA JIA display a significantly higher frequency of CD4+CD25bright T cells with concomitant higher levels of mRNA FoxP3 in the peripheral blood than ext-OA JIA patients. Furthermore, while numbers of synovial fluid (SF) CD4+CD25bright T cells were equal in both patient groups, pers-OA JIA patients displayed a higher frequency of CD4+CD25int T cells and therefore of CD4+CD25total in the SF than ext-OA JIA patients. Analysis of FoxP3 mRNA levels revealed a high expression in SF CD4+CD25bright T cells of both patient groups and also significant expression of FoxP3 mRNA in the CD4+CD25int T cell population. The CD4+CD25bright cells of both patient groups and the CD4+CD25int cells of pers-OA JIA patients were able to suppress responses of CD25neg cells in vitro. A markedly higher expression of CTLA-4, glucocorticoid-induced TNFR, and HLA-DR on SF CD4+CD25bright T regulatory (Treg) cells compared with their peripheral counterparts suggests that the CD4+CD25+ Treg cells may undergo maturation in the joint. In correlation with this mature phenotype, the SF CD4+CD25bright T cells showed an increased regulatory capacity in vitro compared with peripheral blood CD4+CD25bright T cells. These data suggest that CD4+CD25bright Treg cells play a role in determining the patient’s fate toward either a favorable or unfavorable clinical course of disease.
Antiviral Cytokines Induce Hepatic Expression of the Granzyme B Inhibitors, Proteinase Inhibitor 9 and Serine Proteinase Inhibitor 6
Authors: Barrie Mahmoud B. , Stout Heather W. , Abougergi Marwan S. , Miller Bonnie C. , Thiele Dwain L.
Published: April 2014

Expression of the granzyme B inhibitors, human proteinase inhibitor 9 (PI-9), or the murine orthologue, serine proteinase inhibitor 6 (SPI-6), confers resistance to CTL or NK killing by perforin- and granzyme-dependent effector mechanisms. In light of prior studies indicating that virally infected hepatocytes are selectively resistant to this CTL effector mechanism, the present studies investigated PI-9 and SPI-6 expression in hepatocytes and hepatoma cells in response to adenoviral infection and to cytokines produced during antiviral immune responses. Neither PI-9 nor SPI-6 expression was detected by immunoblotting in uninfected murine or human hepatocytes. Similarly, human Huh-7 hepatoma cells were found to express only very low levels of PI-9 relative to levels detected in perforin- and granzyme-resistant CTL or lymphokine-activated killer cells. Following in vivo adenoviral infection or in vitro culture with IFN-αβ or IFN-γ, SPI-6 expression was induced in murine hepatocytes. Similarly, after culture with IFN-α, induction of PI-9 mRNA and protein expression was observed in human hepatocytes and Huh-7 cells. IFN-γ and TNF-α also induced 4- to 10-fold higher levels of PI-9 mRNA expression in Huh-7 cells, whereas levels of mRNA encoding a related serine proteinase inhibitor, proteinase inhibitor 8, were unaffected by culture of Huh-7 cells with IFN-α, IFN-γ, or TNF-α. These findings indicate that cytokines that promote antiviral cytopathic responses also regulate expression of the cytoprotective molecules, PI-9 and SPI-6, in hepatocytes that are potential targets of CTL and NK effector mechanisms.
Lack of Galactose-α-1,3-Galactose Expression on Porcine Endothelial Cells Prevents Complement-Induced Lysis but Not Direct Xenogeneic NK Cytotoxicity
Authors: Baumann Bettina C. , Forte Pietro , Hawley Robert J. , Rieben Robert , Schneider Mårten K. J. , Seebach Jörg D.
Published: April 2014

The galactose-α-1,3-galactose (αGal) carbohydrate epitope is expressed on porcine, but not human cells, and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models, NAb binding to porcine endothelial cells will likely induce complement activation, lysis, and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts, either by direct recognition of activating molecules on target cells or by FcγRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of αGal protects porcine endothelial cells from NAb/complement-induced lysis, direct xenogeneic NK lysis, NAb-dependent ADCC, and adhesion of human NK cells under shear stress. Homologous recombination, panning, and limiting dilution cloning were used to generate an αGal-negative porcine endothelial cell line, PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the αGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However, direct xenogeneic lysis of PED2*3.51, mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92, was not reduced. Furthermore, adhesion of IL-2-activated human NK cells did not rely on αGal expression. In conclusion, removal of αGal leads to a clear reduction in complement-induced lysis and ADCC, but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity, indicating that αGal is not a dominant target for direct human NK cytotoxicity against porcine cells.
Cutting Edge: NTB-A Activates NK Cells via Homophilic Interaction
Authors: Flaig Ruediger M. , Stark Sebastian , Watzl Carsten
Published: April 2014

NK cells are an important component of the innate immune system. Their activity is tightly regulated by activating and inhibitory surface receptors. However, the exact functions of many activating surface receptors, as well as their ligands, still remain to be elucidated. NTB-A is a receptor on the surfaces of human NK, T, and B cells, mediating a signal whose malfunction may be involved in X-linked lymphoproliferative disease. However, the ligand of NTB-A has remained elusive so far. Using trimeric recombinant proteins, we now show that NTB-A is its own ligand. Homophilic interaction of NTB-A enhances NK cell cytotoxicity and influences NK cell proliferation and IFN-γ secretion. We suggest that NTB-A is an interlymphocyte signaling molecule, which serves to orchestrate the activities of immune cells.
Cutting Edge: Effector Memory CD8+ T Cells Play a Prominent Role in Recall Responses to Secondary Viral Infection in the Lung
Authors: Roberts Alan D. , Woodland David L.
Published: April 2014

The relative contributions of CD62Lhigh (central) memory and CD62Llow (effector) memory T cell populations to recall responses are poorly understood, especially in the respiratory tract. In this study, we took advantage of a dual-adoptive transfer system in the mouse to simultaneously follow the recall response of effector and central memory subpopulations to intranasal parainfluenza virus infection. Using MHC class I and class II multimers, we tracked the responses of Ag-specific CD8+ and CD4+ memory T cells in the same animals. The data show that effector memory T cells mounted recall responses that were equal to, or greater than, those mounted by central memory T cells. Moreover, effector memory T cells were more efficient at subsequently establishing a second generation of memory T cells. These data contrast with other studies indicating that central memory CD8+ T cells are the prominent contributors to systemic virus infections.
Small Interfering RNAs Mediate Sequence-Independent Gene Suppression and Induce Immune Activation by Signaling through Toll-Like Receptor 3
Authors: Karikó Katalin , Bhuyan Prakash , Capodici John , Weissman Drew
Published: April 2014

Small interfering (si) and short hairpin (sh) RNAs induce robust degradation of homologous mRNAs, making them a potent tool to achieve gene silencing in mammalian cells. Silencing by siRNAs is used widely because it is considered highly specific for the targeted gene, although a recent report suggests that siRNA also induce signaling through the type I IFN system. When human embryonic kidney 293 (HEK293) or keratinocyte (HaCaT) cell lines or human primary dendritic cells or macrophages were transfected with siRNA or shRNAs, suppression of nontargeted mRNA expression was detected. Additionally, siRNA and shRNA, independent of their sequences, initiated immune activation, including IFN-α and TNF-α production and increased HLA-DR expression, in transfected macrophages and dendritic cells. The siRNAs induced low, but significant, levels of IFN-β in HEK293 and HaCaT cells. Secretion of these cytokines increased tremendously when HEK293 cells overexpressed Toll-like receptor 3 (TLR3), and the increased secretion of IFN-β was inhibited by coexpression of an inhibitor of TIR domain-containing adapter-inducing IFN-β, the TLR3 adaptor protein linked to IFN regulatory factor 3 signaling. Although siRNA and shRNA knockdown of genes represents a new and powerful tool, it is not without nonspecific effects, which we demonstrate are mediated in part by signaling through TLR3.
A Spontaneously Arising Pancreatic Tumor Does Not Promote the Differentiation of Naive CD8+ T Lymphocytes into Effector CTL
Authors: Lyman Michael A. , Aung Sandra , Biggs Judith A. , Sherman Linda A.
Published: April 2014

In this report, we address whether a growing tumor provides sufficient inflammatory signals to promote activation, clonal expansion, and acquisition of effector functions by naive tumor-specific CD8+ T lymphocytes. CD8+ T lymphocytes obtained from hemagglutinin (HA)-specific clone 4 TCR-transgenic mice were injected into recipient mice that spontaneously develop pancreatic tumors expressing HA as a tumor-associated Ag (RIP-Tag2-HA mice). When 3 × 106 clone 4 CD8+ T cells were transferred into tumor-bearing mice, the cells became activated in the pancreatic lymph nodes where they proliferated and acquired effector functions such as cytolytic activity and IFN-γ production. Surprisingly, reducing the number of adoptively transferred CD8+ T cells led to a parallel reduction in the proportion of the activated cells that exhibited effector functions, suggesting that CTL differentiation was induced by the large numbers of activated CD8+ T cells and not the tumor environment. Provision of tumor-specific CD4+ helper cells provided the signals required to promote both the development of CTL effector functions and increased clonal expansion, resulting in tumor eradication. Considering that only small numbers of tumor-specific CD8+ T cells would be present in a conventional T cell repertoire, these data suggest that tumor growth alone may not provide the inflammatory signals necessary to support the development of CD8+ T cell effector functions.
Tolerance to DNA in (NZB × NZW)F1 Mice That Inherit an Anti-DNA VH as a Conventional μ H Chain Transgene but Not as a VH Knock-in Transgene
Authors: Steeves Meredith A. , Marion Tony N.
Published: April 2014

Lupus-prone (NZB × NZW)F1 (BWF1) mice were made transgenic (Tg) for an anti-DNA Ab inherited either as a conventional VH3H9-μ H chain Tg (3H9-μ) with or without a conventional Vκ8-κ Tg, or a VH3H9 VH knock-in Tg allele (3H9R) with or without a Vκ4 Vκ knock-in Tg allele (Vκ4R). VH3H9 yields an anti-DNA Ab with most L chains including an anti-ssDNA with the Vκ8 Tg and an anti-dsDNA with the Vκ4 Tg. BWF1 mice that inherited the conventional 3H9-μ had normal serum IgM, little to none of which was encoded by 3H9-μ, and only a small percentage of those mice had serum anti-DNA, none of which was transgene encoded. B cells expressing the conventional 3H9-μ Tg were anergic. BWF1 mice that inherited the knock-in 3H9R Tg allele also had normal serum IgM, one-half of which was encoded by 3H9R, and produced anti-DNA encoded by the Tg allele. Most B cells expressing the knock-in 3H9R Tg also had an anergic phenotype. The results indicate that autoimmune-prone BWF1 mice initially develop effective B cell tolerance to DNA through anergy, and anergy was sustained in 3H9-μ Tg peripheral B cells but not in 3H9R Tg B cells. B cells expressing the 3H9R knock-in Tg allele were able to achieve an activation threshold that B cells expressing the 3H9-μ conventional Tg could not. The maintenance of B cell tolerance to DNA in autoimmune-prone BWF1 mice appears to differ from both normal mice and autoimmune-prone MRLlpr/lpr mice.