bluuebunny/crossref_metadata_2025 huggingface dataset:
CCR3 Expression Induced by IL-2 and IL-4 Functioning as a Death Receptor for B Cells
Authors: Jinquan Tan , Jacobi Henrik H. , Jing Chen , Millner Anders , Sten Eva , Hviid Lars , Anting Liu , Ryder Lars P. , Glue Christian , Skov Per S. , Jarman Elizabeth , Lamberth Kasper , Malling Hans-Jørgen , Poulsen Lars K.
Published: April 2014

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (∼98% CCR3+). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19+ (∼40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.
Synoviocyte-Mediated Expansion of Inflammatory T Cells in Rheumatoid Synovitis Is Dependent on CD47-Thrombospondin 1 Interaction
Authors: Vallejo Abbe N. , Yang Hongyu , Klimiuk Piotr A. , Weyand Cornelia M. , Goronzy Jörg J.
Published: April 2014

Fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis elicit spontaneous proliferation of autologous T cells in an HLA-DR and CD47 costimulation-dependent manner. T cell costimulation through CD47 is attributed to specific interaction with thrombospondin-1 (TSP1), a CD47 ligand displayed on FLS. CD47 binding by FLS has broad biological impact that includes adhesion and the triggering of specific costimulatory signals. TSP1+ FLS are highly adhesive to T cells and support their aggregation and growth in situ. Long-term cultures of T cells and FLS form heterotypic foci that are amenable to propagation without exogenous growth factors. T cell adhesion and aggregate formation on TSP1+ FLS substrates are inhibited by CD47-binding peptides. In contrast, FLS from arthroscopy controls lack adhesive or T cell growth-promoting activities. CD47 stimulation transduces a costimulatory signal different from that of CD28, producing a gene expression profile that included induction of ferritin L chain, a component of the inflammatory response. Ferritin L chain augments CD3-induced proliferation of T cells. Collectively, these results demonstrate the active role of FLS in the recruitment, activation, and expansion of T cells in a CD47-dependent manner. Because TSP1 is abundantly expressed in the rheumatoid synovium, CD47-TSP1 interaction is proposed to be a key component of an FLS/T cell regulatory circuit that perpetuates the inflammatory process in the rheumatoid joint.
Identifying MHC Class I Epitopes by Predicting the TAP Transport Efficiency of Epitope Precursors
Authors: Peters Björn , Bulik Sascha , Tampe Robert , van Endert Peter M. , Holzhütter Hermann-Georg
Published: April 2014

We are able to make reliable predictions of the efficiency with which peptides of arbitrary lengths will be transported by TAP. The pressure exerted by TAP on Ag presentation thus can be assessed by checking to what extent MHC class I (MHC-I)-presented epitopes can be discriminated from random peptides on the basis of predicted TAP transport efficiencies alone. Best discriminations were obtained when N-terminally prolonged epitope precursor peptides were included and the contribution of the N-terminal residues to the score were down-weighted in comparison with the contribution of the C terminus. We provide evidence that two factors may account for this N-terminal down-weighting: 1) the uncertainty as to which precursors are used in vivo and 2) the coevolution in the C-terminal sequence specificities of TAP and other agents in the pathway, which may vary among the various MHC-I alleles. Combining predictions of MHC-I binding affinities with predictions of TAP transport efficiency led to an improved identification of epitopes, which was not the case when predictions of MHC-I binding affinities were combined with predictions of C-terminal cleavages made by the proteasome.
RNA Interference Shows Critical Requirement for NF-κB p50 in the Production of IL-12 by Human Dendritic Cells
Authors: Laderach Diego , Compagno Daniel , Danos Olivier , Vainchenker William , Galy Anne
Published: April 2014

Specific NF-κB/Rel proteins regulate murine dendritic cell (DC) survival, differentiation, and activation, but little is known of their role in human cells because of limited loss-of-function analyses. RNA interference (RNAi) is a mechanism to effectively silence gene expression via sequence-specific double-stranded small interfering RNAs (siRNAs). RNAi was used to assess the role of the p50 (NF-κB1) protein in the maturation and activation of cultured human monocyte-derived DC (MoDC). Transfection of cultured MoDC with siRNAs reduced p50 mRNA and protein levels in a specific, dose-dependent, and time-dependent manner. Basal or maturation-induced expression of HLA-DR and costimulatory molecules were not affected, whereas transcription of the IL-12 p40 gene and the secretion of IL-12αβ were reduced. Such MoDC induced less IFN-γ production by T cells in MLR. This is the first report of RNAi-induced phenotype in human primary DC with a method that caused no measurable toxicity or type-I IFN response. siRNAs appear useful for the study of signaling pathways in immune cells, revealing a pivotal requirement for p50 in MoDC for IL-12 production and induction of optimal type-1 immune responses.
Incomplete Activation of CD4 T Cells by Antigen-Presenting Transitional Immature B Cells: Implications for Peripheral B and T Cell Responsiveness
Authors: Chung James B. , Wells Andrew D. , Adler Scott , Jacob Anand , Turka Laurence A. , Monroe John G.
Published: April 2014

B cells leave the bone marrow as transitional B cells. Transitional B cells represent a target of negative selection and peripheral tolerance, both of which are abrogated in vitro by mediators of T cell help. In vitro, transitional and mature B cells differ in their responses to B cell receptor ligation. Whereas mature B cells up-regulate the T cell costimulatory molecule CD86 (B7.2) and are activated, transitional B cells do not and undergo apoptosis. The ability of transitional B cells to process and present Ag to CD4 T cells and to elicit protective signals in the absence of CD86 up-regulation was investigated. We report that transitional B cells can process and present Ag as peptide:MHC class II complexes. However, their ability to activate T cells and elicit help signals from CD4-expressing Th cells was compromised compared with mature B cells, unless exogenous T cell costimulation was provided. A stringent requirement for CD28 costimulation was not evident in interactions between transitional B cells and preactivated CD4-expressing T cells, indicating that T cells involved in vivo in an ongoing immune response might rescue Ag-specific transitional B cells from negative selection. These data suggest that during an immune response, immature B cells may be able to sustain the responses of preactivated CD4+ T cells, while being unable to initiate activation of naive T cells. Furthermore, the ability of preactivated, but not naive T cells to provide survival signals to B cell receptor-engaged transitional immature B cells argues that these B cells may be directed toward activation rather than negative selection when encountering Ag in the context of a pre-existing immune response.
KIR2DL4 (CD158d) Genotype Influences Expression and Function in NK Cells
Authors: Goodridge Jodie P. , Witt Campbell S. , Christiansen Frank T. , Warren Hilary S.
Published: April 2014

The expression and function of the NK cell receptor KIR2DL4 are controversial. Two common alleles of the transmembrane domain of KIR2DL4 exist. The 10A allele with 10 adenines at the end of the transmembrane exon encodes a full length receptor, whereas the 9A allele has only 9 adenines resulting in a frame shift which in turn generates a stop codon early in the first cytoplasmic exon. The possibility that the 10A and 9A alleles might result in differences in expression and function of KIR2DL4 was explored using mAbs to KIR2DL4. Transfection experiments with cDNA from the 10A and 9A alleles revealed significant membrane expression only with the protein encoded by the 10A allele. Analysis of peripheral blood NK cells demonstrated that only in subjects with at least one 10A allele was cell surface expression of KIR2DL4 detectable, and then only on the minor CD56bright NK cell subset. The major CD56dim NK cell subset did not cell surface express KIR2DL4 but, interestingly, did so after in vitro culture. Functional analysis using cultured NK cells in redirected lysis assays demonstrated that KIR2DL4 is an activating receptor for NK cells with at least one 10A allele. No significant activity was detected for NK cells generated from subjects homozygous for the 9A allele. These data show that genotype influences cell surface expression and function of KIR2DL4 which may account for reported differences in KIR2DL4 expression and function.
NKT Cells from Normal and Tumor-Bearing Human Livers Are Phenotypically and Functionally Distinct from Murine NKT Cells
Authors: Kenna Tony , Mason Lucy Golden , Porcelli Steven A. , Koezuka Yasuhiko , Hegarty John E. , O’Farrelly Cliona , Doherty Derek G.
Published: April 2014

A major group of murine NK T (NKT) cells express an invariant Vα14Jα18 TCR α-chain specific for glycolipid Ags presented by CD1d. Murine Vα14Jα18+ account for 30–50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Vα24Vβ11+ NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3+ cells) and blood (0.02%). In contrast to those in blood, most hepatic Vα24+ NKT cells express the Vβ11 chain. They include CD4+, CD8+, and CD4−CD8− cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Vα24+ T cells are potent producers of IFN-γ and TNF-α, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, α-galactosylceramide. Vα24+Vβ11+ cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-γ in response to α-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.
Induced Dendritic Cell Differentiation of Chronic Myeloid Leukemia Blasts Is Associated with Down-Regulation of BCR-ABL
Authors: Lindner Inna , Kharfan-Dabaja Mohamed A. , Ayala Ernesto , Kolonias Despina , Carlson Louise M. , Beazer-Barclay Yasmin , Scherf Uwe , Hnatyszyn James H. , Lee Kelvin P.
Published: April 2014

Although differentiation of leukemic blasts to dendritic cells (DC) has promise in vaccine strategies, the mechanisms underlying this differentiation and the differences between leukemia and normal progenitor-derived DC are largely undescribed. In the case of chronic myeloid leukemia (CML), understanding the relationship between the induction of DC differentiation and the expression of the BCR-ABL oncogene has direct relevance to CML biology as well as the development of new therapeutic approaches. We now report that direct activation of protein kinase C (PKC) by the phorbol ester PMA in the BCR-ABL+ CML cell line K562 and primary CML blasts induced nonterminal differentiation into cells with typical DC morphology (cytoplasmic dendrites), characteristic surface markers (MHC class I, MHC class II, CD86, CD40), chemokine and transcription factor expression, and ability to stimulate T cell proliferation (equivalent to normal monocyte-derived DC). PKC-induced differentiation was associated with down-regulation of BCR-ABL mRNA expression, protein levels, and kinase activity. This down-regulation appeared to be signaled through the mitogen-activated protein kinase pathway. Therefore, PKC-driven differentiation of CML blasts into DC-like cells suggests a potentially novel strategy to down-regulate BCR-ABL activity, yet raises the possibility that CML-derived DC vaccines will be less effective in presenting leukemia-specific Ags.
Either of the CD45RB and CD45RO Isoforms Are Effective in Restoring T Cell, But Not B Cell, Development and Function in CD45-Null Mice
Authors: Ogilvy Sarah , Louis-Dit-Sully Christine , Cooper Joanne , Cassady Robin L. , Alexander Denis R. , Holmes Nick
Published: April 2014

The protein tyrosine phosphatase CD45 is expressed as a series of isoforms whose tissue and differentiation stage specificity is broadly conserved in evolution. CD45 has been shown to be an important regulator of a variety of functions in many different hemopoietic lineages. We have chosen an in vivo genetic complementation strategy to investigate the differential functions between isoforms. In this study, we report the characterization of transgenic mice which express the isoforms CD45RO or CD45RB as their only CD45 molecules, at a variety of expression levels and in the majority of hemopoietic lineages. Both CD45RO and CD45RB isoforms reconstitute thymocyte development in a CD45-null mouse background when expressed above a threshold level. The resulting mature T cells populate the peripheral lymphoid organs where they are found at normal frequency. Both CD45RO and CD45RB isoforms also permit T cell function in the periphery, although the threshold for normal function here appears to be set higher than in the thymus. In contrast, neither isoform is capable of fully restoring peripheral B cell maturation, even at levels approaching those in heterozygous CD45+/− mice in which maturation is normal. In vitro activation of B cells by Ag-receptor stimulation is only minimally complemented by these CD45RO and CD45RB transgenes. Our results suggest that CD45 isoforms play unique roles which differ between the T and B lineages.
Break of Neonatal Th1 Tolerance and Exacerbation of Experimental Allergic Encephalomyelitis by Interference with B7 Costimulation
Authors: Bell J. Jeremiah , Min Booki , Gregg Randal K. , Lee Hyun-Hee , Zaghouani Habib
Published: April 2014

Ig-PLP1 is an Ig chimera expressing proteolipid protein-1 (PLP1) peptide corresponding to aa residues 139–151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals comprise Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-γ or IL-4, secrete significant amounts of IL-2. In this work, studies were performed to determine whether costimulation through B7 molecules plays any role in the unusual form of splenic Th1 anergy. The results show that engagement of either B7.1 or B7.2 with anti-B7 Abs during induction of EAE in adult mice that were neonatally tolerized with Ig-PLP1 restores and exacerbates disease severity. At the cellular level, the anergic splenic T cells regain the ability to proliferate and produce IFN-γ when stimulated with Ag in the presence of either anti-B7.1 or anti-B7.2 Ab. However, such restoration was abolished when both B7.1 and B7.2 molecules were engaged simultaneously, indicating that costimulation is necessary for reactivation. Surprisingly, both anti-B7.1 and anti-B7.2 Abs triggered splenic dendritic cells to produce IL-12, a key cytokine required for restoration of the anergic T cells. Thus, recovery from neonatally induced T cell anergy requires B7 molecules to serve double functions, namely, costimulation and induction of cytokine production by APCs.
IL-18 Stimulates the Proliferation and IFN-γ Release of CD4+ T Cells in the Chicken: Conservation of a Th1-Like System in a Nonmammalian Species
Authors: Göbel Thomas W. , Schneider Kirsten , Schaerer Beatrice , Mejri Iris , Puehler Florian , Weigend Steffen , Staeheli Peter , Kaspers Bernd
Published: April 2014

The phylogeny of Th1 and Th2 subsets has not been characterized mainly due to the limited information regarding cytokines in nonmammalian vertebrates. In this study, we characterize a Th1-like regulatory system focusing on the IL-18-regulated IFN-γ secretion. Stimulation of splenocytes with chicken IL-18 induced high levels of IFN-γ secretion. Depletion of either macrophages or CD4+ T cells from the splenocyte cultures caused unresponsiveness to IL-18. In contrast, PBL were unresponsive to IL-18 in the presence or absence of macrophages, but IFN-γ secretion was stimulated by suboptimal anti-TCR cross-linking combined with IL-18. Splenocytes from five different chicken lines responded equally well to the IL-18 treatment. LSL chicken splenocytes, however, responded only to IL-18 when stimulated either with optimal TCR cross-linking alone or suboptimal TCR cross-linking combined with IL-18. IL-18 not only induced IFN-γ secretion, but also stimulated splenocyte proliferation. This IL-18-induced proliferation was compared with the effects observed with IL-2. Both cytokines activated the splenocytes as demonstrated by increased size and MHC class II Ag up-regulation in the case of IL-18. Phenotypic analyses following 6 days of culture revealed that IL-2 mainly affected the proliferation of CD8+ cells, whereas IL-18 had an opposite effect and stimulated the proliferation of CD4+ cells. Taken together, these results demonstrate the conservation of Th1-like proinflammatory responses in the chicken; they characterize IL-18 as a major growth factor of CD4+ T cells and identify two distinct mechanisms of IL-18-induced IFN-γ secretion.
T Cell-Intrinsic Requirement for NF-κB Induction in Postdifferentiation IFN-γ Production and Clonal Expansion in a Th1 Response
Authors: Corn Radiah A. , Aronica Mark A. , Zhang Fuping , Tong Yingkai , Stanley Sarah A. , Kim Se Ryoung Agnes , Stephenson Linda , Enerson Ben , McCarthy Susan , Mora Ana , Boothby Mark
Published: April 2014

NF-κB/Rel transcription factors are linked to innate immune responses and APC activation. Whether and how the induction of NF-κB signaling in normal CD4+ T cells regulates effector function are not well-understood. The liberation of NF-κB dimers from inhibitors of κB (IκBs) constitutes a central checkpoint for physiologic regulation of most forms of NF-κB. To investigate the role of NF-κB induction in effector T cell responses, we targeted inhibition of the NF-κB/Rel pathway specifically to T cells. The Th1 response in vivo is dramatically weakened when T cells defective in their NF-κB induction (referred to as IκBα(ΔN) transgenic cells) are activated by a normal APC population. Analyses in vivo, and IL-12-supplemented T cell cultures in vitro, reveal that the mechanism underlying this T cell-intrinsic requirement for NF-κB involves activation of the IFN-γ gene in addition to clonal expansion efficiency. The role of NF-κB in IFN-γ gene expression includes a modest decrease in Stat4 activation, T box expressed in T cell levels, and differentiation efficiency along with a more prominent postdifferentiation step. Further, induced expression of Bcl-3, a trans-activating IκB-like protein, is decreased in T cells as a consequence of NF-κB inhibition. Together, these findings indicate that NF-κB induction in T cells regulates efficient clonal expansion, Th1 differentiation, and IFN-γ production by Th1 lymphocytes at a control point downstream from differentiation.
The Human Fascin Gene Promoter Is Highly Active in Mature Dendritic Cells Due to a Stage-Specific Enhancer
Authors: Bros Matthias , Ross Xiao-Lan , Pautz Andrea , Reske-Kunz Angelika B. , Ross Ralf
Published: April 2014

Dendritic cells (DC), regarded as the most efficient APCs of the immune system, are capable of activating naive T cells. Thus, DC are primary targets in immunotherapy. However, little is known about gene regulation in DC, and for efficient transcriptional targeting of human DC, a suitable promoter is still missing. Recently, we successfully used the promoter of the murine actin-bundling protein fascin to transcriptionally target DC by DNA vaccination in mice. In this study, we report on isolation of the human fascin promoter and characterization of its regulatory elements. The actively expressed gene was distinguished from a conserved inactive genomic locus and a continuous region of 14 kb covering the gene and 3 kb of 5′-flanking sequences was subcloned, sequenced, and analyzed for regulatory elements. Regulatory sequences were found solely in the 5′-flanking promoter region. The promoter exerted robust activity in DC and a fascin-positive neuronal cell line, but not in the fascin-negative cells tested. Notably, promoter activity in DC markedly increased with maturation of DC. By progressive 5′ deletion, we identified a core promoter region, harboring a putative GC box, a composite cAMP responsive element/AP-1 binding site and a TATA box. By internal deletion, we demonstrated functional importance of either regulatory element. Furthermore, we identified a more distal stage-specific enhancer region also containing silencer elements. Taken together, the human fascin promoter allows for transcriptional targeting of mature DC and represents a promising tool for immunotherapy. To our knowledge, this study reports for the first time on promoter activity in human monocyte-derived DC.
Inhibitory Coreceptors Activated by Antigens But Not by Anti-Ig Heavy Chain Antibodies Install Requirement of Costimulation Through CD40 for Survival and Proliferation of B Cells
Authors: Hokazono Yasuhisa , Adachi Takahiro , Wabl Matthias , Tada Nobuhiko , Amagasa Teruo , Tsubata Takeshi
Published: April 2014

Ag-induced B cell proliferation in vivo requires a costimulatory signal through CD40, whereas B cell Ag receptor (BCR) ligation by anti-Ig H chain Abs, such as anti-Ig μ H chain Ab and anti-Ig δ H chain Ab, alone induces proliferation of B cells in vitro, even in the absence of CD40 ligation. In this study, we demonstrate that CD40 signaling is required for survival and proliferation of B cells stimulated by protein Ags in vitro as well as in vivo. This indicates that the in vitro system represents B cell activation in vivo, and that protein Ags generate BCR signaling distinct from that by anti-Ig H chain Abs. Indeed, BCR ligation by Ags, but not by anti-Ig H chain Abs, efficiently phosphorylates the inhibitory coreceptors CD22 and CD72. When these coreceptors are activated, anti-Ig H chain Ab-stimulated B cells can survive and proliferate only in the presence of CD40 signaling. Conversely, treatment of Ag-stimulated B cells with anti-CD72 mAb blocks CD72 phosphorylation and induces proliferation, even in the absence of CD40 signaling. These results strongly suggest that activation of B cells by anti-Ig H chain Abs involves their ability to silence the inhibitory coreceptors, and that the inhibitory coreceptors install requirement of CD40 signaling for survival and proliferation of Ag-stimulated B cells.
α3 Domain Mutants of Peptide/MHC Class I Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells
Authors: Pittet Mikaël J. , Rubio-Godoy Verena , Bioley Gilles , Guillaume Philippe , Batard Pascal , Speiser Daniel , Luescher Immanuel , Cerottini Jean-Charles , Romero Pedro , Zippelius Alfred
Published: April 2014

The goal of adoptive T cell therapy in cancer is to provide effective antitumor immunity by transfer of selected populations of tumor Ag-specific T cells. Transfer of T cells with high TCR avidity is critical for in vivo efficacy. In this study, we demonstrate that fluorescent peptide/MHC class I multimeric complexes incorporating mutations in the α3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor can be used to selectively isolate tumor Ag-specific T cells of high functional avidity from both in vitro expanded and ex vivo T cell populations. Sorting, cloning, and expansion of α3 domain mutant multimer-positive CD8 T cells enabled rapid selection of high avidity tumor-reactive T cell clones. Our results are relevant for ex vivo identification and isolation of T cells with potent antitumor activity for adoptive T cell therapy.
Reduced Dosage of Bruton’s Tyrosine Kinase Uncouples B Cell Hyperresponsiveness from Autoimmunity inlyn−/− Mice
Authors: Whyburn Lindsey R. , Halcomb Kristina E. , Contreras Cristina M. , Lowell Clifford A. , Witte Owen N. , Satterthwaite Anne B.
Published: April 2014

The development of autoimmunity is correlated with heightened sensitivity of B cells to B cell Ag receptor (BCR) cross-linking. BCR signals are down-regulated by Lyn, which phosphorylates inhibitory receptors. lyn−/− mice have reduced BCR signaling thresholds and develop autoantibodies, glomerulonephritis, splenomegaly due to myeloid hyperplasia, and increased B-1 cell numbers. Bruton’s tyrosine kinase (Btk), a critical component of BCR signaling pathways, is required for autoantibody production in lyn−/− mice. It is unclear whether Btk mediates autoimmunity at the level of BCR signal transduction or B cell development, given that lyn−/−Btk−/− mice have a severe reduction in conventional B and B-1 cell numbers. To address this issue, we crossed a transgene expressing a low dosage of Btk (Btklow) in B cells to lyn−/−Btk−/− mice. Conventional B cell populations were restored to levels similar to those in lyn−/− mice. These cells were as hypersensitive to BCR cross-linking as lyn−/− B cells as measured by proliferation, Ca2+ flux, and activation of extracellular signal-regulated kinase and Akt. However, lyn−/−Btklow mice did not produce anti-ssDNA, anti-dsDNA, anti-histone, or anti-histone/DNA IgM or IgG. They also lacked B-1 cells and did not exhibit splenomegaly. Thus, B cell hyperresponsiveness is insufficient for autoimmunity in lyn−/− mice. These studies implicate B-1 and/or myeloid cells as key contributors to the lyn−/− autoimmune phenotype.
Apoptosis by Neglect of CD4+ Th Cells in Granulomas: A Novel Effector Mechanism Involved in the Control of Egg-Induced Immunopathology in Murine Schistosomiasis
Authors: Rutitzky Laura I. , Mirkin Gerardo A. , Stadecker Miguel J.
Published: April 2014

In infection with Schistosoma mansoni, parasite eggs precipitate an intrahepatic granulomatous and fibrosing inflammation that is mediated by CD4+ Th cells. Compared with CBA mice, C57BL/6 mice develop smaller granulomas composed of cells that exhibit reduced proliferative responses to schistosome egg Ags. In the present study, we investigated CD4+ T cell apoptosis as a possible mechanism that could account for this subdued response. We found throughout the course of several infection weeks a markedly higher proportion of apoptotic CD4+ T cells in granulomas from C57BL/6 mice than in those from CBA mice ex vivo; the apoptosis further increased upon cell cultivation in vitro. Activation-induced cell death or CD8+ T cells failed to account for the enhanced apoptosis as infected Fas-, Fas ligand,- and CD8-deficient mice exhibited similar apoptosis to that seen in wild-type counterparts. However, a strikingly lower IL-2 production by schistosome egg Ag-stimulated C57BL/6 granuloma and mesenteric lymph node cells suggested the possibility of apoptosis due to growth factor deprivation. Indeed, the CD4+ T cell apoptosis was significantly reversed by addition of rIL-2 in vitro, or by injection of rIL-2 in vivo, which also resulted in significant exacerbation of granulomatous inflammation. These findings indicate that apoptosis by neglect can represent a significant means of controlling CD4+ T cells that mediate the immunopathology in schistosomiasis.
Pathogen-Associated Molecular Patterns Sensitize Macrophages to Fas Ligand-Induced Apoptosis and IL-1β Release
Authors: Fukui Masayuki , Imamura Ryu , Umemura Masayuki , Kawabe Takaya , Suda Takashi
Published: April 2014

Antigenic stimulation activates T cells and simultaneously destines them to die by Fas-mediated apoptosis. In this study, we demonstrated that various pathogen-associated molecular patterns up-regulated Fas expression in macrophages and sensitized them specifically to Fas ligand (FasL), but not to other apoptosis-inducing agents such as TNF-α, etoposide (VP-16), and staurosporine. Toll-like receptor, NF-κB, and p38 mitogen-activated protein kinase mediated these responses. LPS stimulation induced the expression of Fas, caspase 8, cellular FLIP Bfl-1/A1, and Bcl-x, but not FasL, TNFR p55, Bak, Bax, and Bad at the transcriptional level. Thus, LPS selectively induced the expression of apoptotic molecules of the Fas death pathway (except for cellular FLIP) and antiapoptotic molecules of the mitochondrial death pathway. However, the kinetics of macrophage disappearance following Escherichia coli-induced peritonitis was similar between wild-type and Fas-deficient mice, suggesting that Fas is not essential for the turnover of activated macrophages in T cell-independent inflammation. In contrast, LPS-activated macrophages produced a large amount of IL-1β upon FasL stimulation. Thus, unlike the activation-induced cell death of T cells, the sensitization of macrophages to FasL by pathogen-associated molecular patterns seems to be a proinflammatory rather than an anti-inflammatory event.
A Catalytically Inactive Form of Protein Kinase C-Associated Kinase/Receptor Interacting Protein 4, a Protein Kinase Cβ-Associated Kinase That Mediates NF-κB Activation, Interferes with Early B Cell Development
Authors: Cariappa Annaiah , Chen Luojing , Haider Khaleda , Tang Mei , Nebelitskiy Eugene , Moran Stewart T. , Pillai Shiv
Published: April 2014

Protein kinase C-associated kinase (PKK)/receptor interacting protein 4 (RIP4) is a protein kinase C (PKC) β-associated kinase that links PKC to NF-κB activation. The kinase domain of PKK is similar to that of RIP, RIP2, and RIP3. We show in this study that PKK is expressed early during lymphocyte development and can be detected in common lymphoid progenitor cells. Targeting of a catalytically inactive version of PKK to lymphoid cells resulted in a marked impairment in pro-B cell generation in the bone marrow. Although peripheral B cell numbers were markedly reduced, differentiation into follicular and marginal zone B cells was not defective in these mice. B-1a and B-1b B cells could not be detected in these mice, but this might be a reflection of the overall defect in B cell production observed in these animals. In keeping with a possible link to PKCβ, peripheral B cells in these mice exhibit a defect in anti-IgM-mediated proliferation. These studies suggest that PKK may be required early in B cell development and for BCR-mediated B cell proliferation.
Studies of Structure-Activity Relations of Complement Inhibitor Compstatin
Authors: Soulika Athena M. , Morikis Dimitrios , Sarrias Maria-Rosa , Roy Melinda , Spruce Lynn A. , Sahu Arvind , Lambris John D.
Published: April 2014

Compstatin, a 13-mer cyclic peptide, is a novel and promising inhibitor of the activation of the complement system. In our search for a more active analog and better understanding of structure-functions relations, we designed a phage-displayed random peptide library based on previous knowledge of structure activity relations, in which seven amino acids deemed necessary for structure and activity were kept fixed while the remaining six were optimized. Screening of this library against C3 identified four binding clones. Synthetic peptides corresponding to these clones revealed one analog, called acetylated Ile1Leu/His9Trp/Thr13Gly triple replacement analog of compstatin corresponding to clone 640 (Ac-I1L/H9W/T13G), which was more active than compstatin. This newly identified peptide had 4-fold higher activity when compared with the originally isolated form of compstatin and 1.6-fold higher activity when compared with acetylated compstatin (Ac-compstatin). The structures of Ac-I1L/H9W/T13G and Ac-compstatin were studied by nuclear magnetic resonance, compared with the structure of compstatin, and found to be very similar. The binding of Ac-I1L/H9W/T13G and the equally active acetylated analog with His9Ala replacement (Ac-H9A) to C3 was evaluated by surface plasmon resonance, which suggested similarity in their binding mechanism but difference when compared with Ac-compstatin. Compensatory effects of flexibility outside the β-turn and tryptophan ring stacking may be responsible for the measured activity increase in Ac-I1L/H9W/T13G and acetylated analog with His9Ala replacement and the variability in binding mechanism compared with Ac-compstatin. These data demonstrate that tryptophan is a key amino acid for activity. Finally, the significance of the N-terminal acetylation was examined and it was found that the hydrophobic cluster at the linked termini of compstatin is essential for binding to C3 and for activity.
New Programming of IL-4 Receptor Signal Transduction in Activated T Cells: Stat6 Induction and Th2 Differentiation Mediated by IL-4Rα Lacking Cytoplasmic Tyrosines
Authors: Mora Ana L. , Stephenson Linda M. , Enerson Ben , Youn Jeehee , Keegan Achsah D. , Boothby Mark
Published: April 2014

Signaling by the IL-4 receptor α-chain (IL-4Rα) is a key determinant of the development of the Th2 lineage of effector T cells. Studies performed in tissue culture cell lines have indicated that tyrosines of the IL-4Rα cytoplasmic tail are necessary for the induction of Stat6, a transcription factor required for Th2 differentiation. Surprisingly, we have found that in activated T cells, IL-4Rα chains lacking all cytoplasmic tyrosines promote induction of this IL-4-specific transcription factor and efficient commitment to the Th2 lineage. Mutagenesis of a tyrosine-free cytoplasmic tail identifies a requirement for the serine-rich ID-1 region in this new program of IL-4R signal transduction observed in activated T cells. Additional findings suggest that an extracellular signal-regulated kinase pathway can be necessary and sufficient for the ability of such tyrosine-free IL-4Rα chains to mediate Stat6 induction. These results provide novel evidence that the molecular mechanisms by which a cytokine specifically induces a Stat transcription factor can depend on the activation state of T lymphoid cells. Furthermore, the data suggest that one pathway by which such new programming may be achieved is mediated by extracellular signal-regulated mitogen-activated protein kinases.
The CD43 Coreceptor Molecule Recruits the ζ-Chain as Part of Its Signaling Pathway
Authors: Cruz-Muñoz Mario Ernesto , Salas-Vidal Enrique , Salaiza-Suazo Norma , Becker Ingeborg , Pedraza-Alva Gustavo , Rosenstein Yvonne
Published: April 2014

CD43 is an abundant cell surface sialoglycoprotein implicated in hemopoietic cell adhesion and activation. Cell stimulation through CD43 results in recruitment of different signaling proteins, including members of the Src family kinases, Syk, phospholipase Cγ2, the adapter protein Shc, the guanine nucleotide exchange factor Vav, and activation of protein kinase C. In this study, we report that in human T lymphocytes, the ζ-chain is part of the CD43 signaling pathway. Upon CD43 engagement, the ζ-chain was tyrosine-phosphorylated, generating docking sites for tyrosine-phosphorylated ζ-associated protein of 70 kDa and Vav. In vitro kinase assays suggested that ζ-associated protein of 70 kDa could account for the kinase activity associated with the ζ-chain following CD43 engagement. Cross-linking CD43 on the surface of the Lck-deficient JCaM.1 cells failed to phosphorylate the ζ-chain and associated proteins, suggesting that Lck is a key element in the CD43 signaling pathway leading to ζ phosphorylation. CD43 engagement with beads coated with anti-CD43 mAb resulted in concentration of the ζ-chain toward the bead attachment site, but interestingly, the distribution of the T cell Ag receptor complex remained unaffected. Recruitment of the ζ-chain through CD43-mediated signals was not restricted to T lymphocytes because phosphorylation and redistribution of the ζ-chain was also observed in NK cells. Our results provide evidence that the ζ-chain functions as a scaffold molecule in the CD43 signaling pathway, favoring the recruitment and formation of downstream signaling complexes involved in the CD43-mediated cell activation of T lymphocytes and other leukocytes such as NK cells.
Potential Role of NKG2D/MHC Class I-Related Chain A Interaction in Intrathymic Maturation of Single-Positive CD8 T Cells
Authors: Hüe Sophie , Monteiro Renato C. , Berrih-Aknin Sonia , Caillat-Zucman Sophie
Published: April 2014

The nonclassical MHC class I molecule MHC class I-related chain A (MICA) interacts with the NKG2D receptor expressed at the surface of most peripheral CD8 T cells, γδ T cells, and NK cells. We investigated the role of MICA-NKG2D interactions in the selection or maturation of the T cell repertoire within the thymus using MICA tetramers and anti-MICA mAbs. MICA tetramers identified a small population of late stage CD8 single-positive, CD45RA+ CD62L+ CCR7+ CD69− thymocytes, a phenotype compatible with that of fully mature CD8+ cells ready to emigrate to the periphery as naive cells. MICA molecules were expressed in the outer layer of Hassal’s corpuscles within the medulla of normal thymus. In thymomas, an overexpression of MICA in cortical and medullar epithelial cells was observed. This was associated with a decreased percentage of NKG2D-positive thymocytes, which expressed a less mature phenotype than in normal thymus. These results indicate that CD8+ thymocytes up-regulate NKG2D as they complete their developmental program before leaving the thymic medulla to seed the periphery, and identify NKG2D as a potential regulator of the developmental processes in T cells that are essential for immune homeostasis.
β2-Microglobulin-Free HLA Class I Heavy Chain Epitope Mimicry by Monoclonal Antibody HC-10-Specific Peptide
Authors: Perosa Federico , Luccarelli Grazia , Prete Marcella , Favoino Elvira , Ferrone Soldano , Dammacco Franco
Published: April 2014

mAb HC-10 loses its reactivity with HLA class I (HLA-I) H chain (HC) following its association with β2-microglobulin (β2m). Furthermore, the HC-10 defined epitope appears to be involved in the pathogenesis of spondyloarthropathies, because HC-10 reduced their incidence in HLA-B27+β2m°/MHC class II knockout mice. This study has characterized the determinant recognized by HC-10. Panning of a phage display peptide library with HC-10 resulted in isolation of the motif PxxWDR, which could be aligned with P57, W60, D61, and R62 of the first domain of the HLA-I HC allospecificities reactive with HC-10. The 55EGPEYWDR(N/E)T64 (p-1) is the shortest motif-bearing peptide that reacts with HC-10 and inhibits its binding to soluble HLA-B7 HC, irrespective of whether N (p-1a) or E (p-1b) is present at position 63. By contrast, HC-10 did not react with six additional peptides, each bearing motif amino acid substitutions present in HC-10-not-reactive HLA-I allospecificities. The p-1-derived Qp-1, synthesized with the additional conserved Q54, which displays the highest in vitro reactivity with HC-10, was the only one to induce in mice IgG resembling HC-10 in their fine specificity. Mapping of the HC-10-defined determinant suggests that the lack of mAb reactivity with β2m-associated HLA-I HC is caused by blocking by the peptide in the groove of β2m-associated HLA-I HC, though a role of HC conformational changes following its association with β2m cannot be excluded. This information contributes to our understanding of the molecular basis of the antigenic profiles of β2m-free and β2m-associated HLA-I HC and may serve to develop active specific immunotherapy of spondyloarthropathies.
A Novel −66T/C Polymorphism in FcεRI α-Chain Promoter Affecting the Transcription Activity: Possible Relationship to Allergic Diseases
Authors: Hasegawa Masanari , Nishiyama Chiharu , Nishiyama Makoto , Akizawa Yushiro , Mitsuishi Kouichi , Ito Tomonobu , Kawada Hiroshi , Furukawa Susumu , Ra Chisei , Okumura Ko , Ogawa Hideoki
Published: April 2014

We found a novel polymorphism, −66T/C, in the promoter region of human FcεRIα, the specific component of the high affinity receptor for IgE (FcεRI), which is essential for the cell surface expression of FcεRI and the binding of IgE Ab. When the effect of the single nucleotide replacement on the promoter function was analyzed, the transcription activity of the T allele promoter was found to be higher than that of the C allele promoter, and was markedly up-regulated by the overexpression of GATA-1 when compared with the C allele promoter. This is probably because the promoter with T at −66 has an additional GATA-1-binding motif in the region, which may assure higher affinity of the transcription factor to the promoter. In accordance with this, EMSA actually indicated that GATA-1 bound to the T allele probe (−80/−59) with the affinity higher than that to the C allele probe. Statistical analysis suggested that a significant portion of nonallergic individuals has heterozygous −66T/C genotype, while most of allergic individuals have homozygous −66T/T genotype in Japanese population. Our findings for the first time demonstrate the presence of FcεRIα polymorphism related to the allergic diseases.
Recombinant TCR Ligand Induces Early TCR Signaling and a Unique Pattern of Downstream Activation
Authors: Wang Chunhe , Mooney Jeffery L. , Meza-Romero Roberto , Chou Yuan K. , Huan Jianya , Vandenbark Arthur A. , Offner Halina , Burrows Gregory G.
Published: April 2014

Recombinant TCR ligands (RTLs) consisting of covalently linked α1 and β1 domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP72–89) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP72–89 peptide-specific CD4+ T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP72–89-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP72–89 peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3ζ p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3ε treatment produced stronger activation of these cellular events with additional activation of NF-κB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-κB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4+ T cells.
Human Immature Monocyte-Derived Dendritic Cells Express the G Protein-Coupled Receptor GPR105 (KIAA0001, P2Y14) and Increase Intracellular Calcium in Response to its Agonist, Uridine Diphosphoglucose
Authors: Skelton Lisa , Cooper Mike , Murphy Marianne , Platt Adam
Published: April 2014

Dendritic cells (DC) are essential to the initiation of an immune response due to their unique ability to take-up and process Ag, translocate to lymph nodes, and present processed Ag to naive T cells. Many chemokines, chemokine receptors and other G protein-coupled receptors (GPCRs) are implicated in these various aspects of DC biology. Through microarray analysis, we compared expression levels of chemokines, their cognate receptors, and selected GPCRs in human monocytes and in vitro monocyte-derived immature and mature DC. Hierarchical clustering of gene expression clearly distinguishes the three cell types, most notably highlighting exceptional levels of expression of the GPCR GPR105 within the immature monocyte-derived DC (MDDC) gene cluster. Little or no expression was observed within the monocyte and mature MDDC cluster. Putative functionality of the GPR105 receptor was demonstrated by an observed calcium flux in immature MDDC treated with the potent GPR105 agonist, uridine 5′-diphosphoglucose (UDP-glucose), while no response to the nucleotide sugar was seen in monocytes and mature MDDC. This UDP-glucose-induced calcium response was, at least in part, pertussis toxin-sensitive. Moreover, immature MDDC from some donors treated with UDP-glucose exhibit an increase in expression of the costimulatory molecule CD86, which correlates with the intensity of the UDP-glucose-induced calcium flux. Together, these data demonstrate differential expression of GPR105 on immature and mature MDDC and suggest a role for the receptor and its agonist ligand in DC activation.
Brugia malayiMicrofilariae Induce Cell Death in Human Dendritic Cells, Inhibit Their Ability to Make IL-12 and IL-10, and Reduce Their Capacity to Activate CD4+ T Cells
Authors: Semnani Roshanak Tolouei , Liu Anne Y. , Sabzevari Helen , Kubofcik Joseph , Zhou Jun , Gilden Julia K. , Nutman Thomas B.
Published: April 2014

Parasite Ag-specific T cell unresponsiveness and diminished IFN-γ production are immunologic hallmarks of patent infection with lymph-dwelling filarial nematodes. Although this diminished responsiveness is directed primarily against the intravascular microfilarial (MF) parasite stage and mediated in part by reduced APC function, the mechanisms involved are not fully understood. In this report, we demonstrate that human dendritic cells (DC) exposed to live MF up-regulate both the cell surface and gene expression of CD54 (ICAM-1). Moreover, live MF result in a 3-fold increase in DC death compared with MF-unexposed DC, primarily due to apoptosis. Notably, microarray and real-time RT-PCR data indicate that live MF concurrently up-regulate mRNA expression of proinflammatory molecules such as IL-8, RANTES, IL-1α, TNF-α, and IL-β in DC, the presence of which is also detected at the protein level, while inhibiting the production of IL-12 (p40 and p70) and IL-10. Soluble excretory-secretory products from live MF diminished IL-12 and IL-10 production and induced DC death, although to a lesser degree. Moreover, exposure of DC to live MF resulted in a decrease in the ability of DC to promote CD4+ T cell production of IFN-γ and IL-5. Our findings clearly suggest that the interaction between live MF and DC is complex but contributes to the hyporesponsiveness and parasite persistence associated with the MF+ state in the infected human. These data further suggest that MF induce an orchestrated response in APC that leads to a diminished capacity to function appropriately, which in turn has significant consequences for CD4+ T cells.