bluuebunny/crossref_metadata_2025 huggingface dataset:
Early Ontogeny of Thymocytes in Pigs: Sequential Colonization of the Thymus by T Cell Progenitors
Authors: Šinkora Marek , Šinkora Jirí , Reháková Zuzana , Butler John E.
Published: April 2014

Successive colonization of the thymus by waves of thymocyte progenitors has been described in chicken-quail chimeras and suggested from studies in mice. In swine, we show that the first CD3ε-bearing thymocytes appear on day 40 of gestation (DG40). These early thymocytes were CD3εhigh and belonged to the γδ T cell lineage. Mature CD3εhigh αβ thymocytes were observed 15 days later (DG55), and their occurrence was preceded by the appearance of CD3εlow thymocytes (DG45). Thereafter, we observed transient changes in thymocyte subset composition (DG56-DG74), which can be explained by a gap in pro-T cell delivery to the thymus. This delivery gap corresponds with the expression of the pan-leukocyte CD45 and pan-myelomonocytic SWC3a markers in fetal liver and bone marrow and is probably caused by shifting of primary lymphopoiesis between these organs. Therefore, we conclude that the embryonic thymus is colonized by at least two successive waves of hemopoietic progenitors during embryogenesis and that the influx of thymocyte progenitors is discontinuous. Surface immunophenotyping and cell cycle analysis of thymocyte subsets allowed us to compare thymocyte differentiation in pigs with that described for rodents and humans and to propose a model for T cell lymphopoiesis in swine. We also observed that the porcine IL-2Rα (CD25), a typical differentiation marker of pre-T cells in mice and humans, was not expressed on thymocyte precursors in pigs and could only be found on mature thymocytes. Finally, we observed a subset of TCRγδ+ thymocytes that were cycling late during their development in the thymus.
Cytokine Production and Killer Activity of NK/T-NK Cells Derived with IL-2, IL-15, or the Combination of IL-12 and IL-18
Authors: Lauwerys Bernard R. , Garot Nathalie , Renauld Jean-Christophe , Houssiau Frédéric A.
Published: April 2014

NK cell populations were derived from murine splenocytes stimulated by IL-2, IL-15, or the combination of IL-12 and IL-18. Whereas NK cells derived with the latter cytokines consisted of an homogeneous population of NK cells (DX5+CD3−), those derived with IL-2 or IL-15 belonged to two different populations, namely NK cells (DX5+CD3−) and T-NK cells (DX5+CD3+). Among NK cells, only those derived with IL-12/IL-18 produced detectable levels of cytokines, namely IFN-γ, IL-10, and IL-13 (with the exception of IL-13 production by NK cells derived with IL-2). As for T-NK cells, IL-2-stimulated cells produced a wide range of cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, but no IFN-γ, whereas IL-15-derived T-NK cells failed to produce any cytokine. Switch-culture experiments indicated that T-NK cells derived in IL-2 and further stimulated with IL-12/IL-18 produced IFN-γ and higher IL-13 levels. Next, we observed that NK/T-NK cell populations exerted distinct effects on Ig production by autologous splenocytes according to the cytokines with which they were derived. Thus, addition of NK cells derived in IL-12/IL-18 inhibited Ig production and induced strong cytotoxicity against splenocytes, whereas addition of NK or T-NK cells grown in IL-2 or IL-15 did not. Experiments performed in IFN-γR knockout mice demonstrated that IFN-γ was not involved in the killer activity of IL-12/IL-18-derived NK cells. The hypothesis that their cytotoxic activity was related to the induction of target apoptosis was confirmed on murine A20 lymphoma cells. Experiments performed in MRL/lpr mice indicated that IL-12/IL-18-derived NK cells displayed their distinct killer activity through a Fas-independent pathway. Finally, perforin was much more expressed in IL-12/IL-18-derived NK cells as compared with IL-2- or IL-15-derived NK cells, an observation that might explain their unique cytotoxicity.
In Vitro and In Vivo Transfection of p21 Gene Enhances Cyclosporin A-Mediated Inhibition of Lymphocyte Proliferation
Authors: Khanna Ashwani K. , Hosenpud Jeffrey D.
Published: April 2014

Cyclosporine has potent antiproliferative properties, some of which may be via the induction of the cyclin inhibitor p21. In this study, we describe the effects of in vitro and in vivo transfection of p21 in lymphoid and nonlymphoid cells. For in vitro studies, p21 sense plasmid DNA was transfected in A-549 cells (lung adenocarcinoma cell line) and Jurkat cells (human lymphoid cell line). This in vitro transfection of p21 resulted in the inhibition of spontaneous and mitogen-induced cellular proliferation ([3H]thymidine uptake) and also augmented the antiproliferative effects of cyclosporine. In vivo transfection of p21 was accomplished in mice via the i.m. injection of p21 sense plasmid DNA complexed with cationic lipids. As was the case in the cell lines, p21 mRNA was augmented in heart, lung, liver, and spleen 7 days after i.m. injection of p21 sense plasmid DNA. The mitogen (anti-CD3)-induced proliferation of splenocytes from p21-overexpressing mice was significantly decreased, and again this effect was augmented by cotreatment with cyclosporine. These novel findings demonstrate the potential of targeting the cell cycle directly to inhibit alloimmune activation in organ transplantation. This may serve as an alternate strategy to induce immunosuppression, perhaps with less toxicity than that which is seen with conventional immunosuppressive agents.
CD81 and CD28 Costimulate T Cells Through Distinct Pathways
Authors: Witherden Deborah A. , Boismenu Richard , Havran Wendy L.
Published: April 2014

We have examined the role of CD81 in the activation of murine splenic αβ T cells. Expression of the CD81 molecule on T cells increases following activation, raising the possibility of a role for this molecule in progression of the activation process. Using an in vitro costimulation assay, we show that CD81 can function as a costimulatory molecule on both CD4+ and CD8+ T cells. This costimulation functions independently of CD28, and unlike costimulation through CD28, is susceptible to inhibition by cyclosporin A. Strikingly, the pattern of cytokine production elicited by costimulation via CD81 is unique. IL-2 production was not up-regulated, whereas both IFN-γ and TNF-α expression significantly increased. Together our results demonstrate an alternate pathway for costimulation of T cell activation mediated by CD81.
Helicobacter pylori Urease Binds to Class II MHC on Gastric Epithelial Cells and Induces Their Apoptosis
Authors: Fan Xuejun , Gunasena Harshani , Cheng Zhijei , Espejo Rosario , Crowe Sheila E. , Ernst Peter B. , Reyes Victor E.
Published: April 2014

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC− cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.
IFN-α and IL-12 Induce IL-18 Receptor Gene Expression in Human NK and T Cells
Authors: Sareneva Timo , Julkunen Ilkka , Matikainen Sampsa
Published: April 2014

IL-18 is a proinflammatory cytokine that enhances innate and specific Th1 immune responses. During microbial infections, IL-18 is produced by activated macrophages. IL-18 exerts its effects in synergy with IFN-α or IL-12 to induce IFN-γ. Here we show that in human NK and T cells IFN-α and IL-12 strongly up-regulate mRNA expression of the IL-18R components, accessory protein-like (AcPL) and IL-1R-related protein (IL-1Rrp). In addition, IFN-α enhanced the expression of MyD88, an adaptor molecule involved in IL-18 signaling. Pretreatment of T cells with IFN-α or IL-12 enhanced IL-18-induced NF-κB activation and sensitized the cells to respond to lower concentrations of IL-18. AcPL and IL-1Rrp genes were strongly expressed in T cells polarized with IL-12, whereas in IL-4-polarized cells these genes were expressed at very low levels, indicating that AcPL and IL-1Rrp genes are preferentially expressed in Th1 cells. In conclusion, the results suggest that IFN-α and IL-12 enhance innate as well as Th1 immune response by inducing IL-18R expression.
Transduction of Dendritic Cells with Bcl-xL Increases Their Resistance to Prostate Cancer-Induced Apoptosis and Antitumor Effect in Mice
Authors: Pirtskhalaishvili Georgi , Shurin Galina V. , Gambotto Andrea , Esche Clemens , Wahl Madeline , Yurkovetsky Zoya R. , Robbins Paul D. , Shurin Michael R.
Published: April 2014

We have shown that prostate cancer (PCa) causes apoptosis of dendritic cells (DC), which might block the development of specific antitumor immune responses. Analysis of murine prostatic carcinoma tissues revealed the significant decrease in intratumoral DC number during tumor progression. We demonstrated that the cytokine-mediated increase in DC survival was accompanied by an elevated expression of the anti-apoptotic protein Bcl-xL. Next, we evaluated the resistance to tumor-induced apoptosis and the antitumor efficiency of genetically engineered DC overexpressing Bcl-xL. DC were transduced with an adenoviral vector encoding the murine Bcl-xL gene and injected intratumorally. Data analysis revealed that treatment of PCa-bearing mice with Bcl-xL-transduced DC resulted in significant inhibition of tumor growth compared with the administration of nontransduced DC. Thus, our data suggest that the protection of DC from PCa-induced apoptosis might significantly increase the efficacy of DC-based therapies in cancer even in the absence of available tumor-specific Ags.
CD11c+ Eosinophils in the Murine Thymus: Developmental Regulation and Recruitment upon MHC Class I-Restricted Thymocyte Deletion
Authors: Throsby Mark , Herbelin André , Pléau Jean-Marie , Dardenne Mireille
Published: April 2014

Eosinophils are bone marrow-derived cells released into the circulation during hypersensitivity reactions and parasitic infections. Under normal conditions most eosinophils are tissue bound, where their physiologic role is unclear. During in situ analysis of the thymic microenvironment for CD11c+ dendritic cell subpopulations (APC critical in the process of thymic negative selection) a discrete population of CD11b/CD11c double-positive cells concentrated in the cortico-medullary region of young mice was detected. Thymic CD11c+ cells were isolated, and the CD11b+ subpopulation (CD44high, class IIlow, CD11cint) was identified as mature eosinophils based on: scatter characteristics, major basic protein mRNA expression, and eosinophilic granules. They are hypodense, release high levels of superoxide anion, and express CD25, CD69, and mRNA for IL-4 and IL-13, but not GM-CSF or IL-5, suggesting a distinct state of activation. Thymic eosinophils are preferentially recruited during the neonatal period; absolute numbers increased 10-fold between 7–14 days to reach parity with dendritic cells before diminishing. In a model of acute negative selection, eosinophil numbers were increased 2-fold 6 h after cognate peptide injection into MHC class I-restricted female H-Y TCR transgenic mice. In both peptide-treated female and negatively selecting male H-Y TCR mice, clusters of apoptotic bodies were associated with eosinophils throughout the thymus. Our data demonstrate a temporal and spatial association between eosinophil recruitment and class I-restricted selection in the thymus, suggesting an immunomodulatory role for eosinophils under nonpathological conditions.
The MHC Class II Molecule I-Ag7 Exists in Alternate Conformations That Are Peptide Dependent
Authors: Arneson Lynne S. , Peterson Mary , Sant Andrea J.
Published: April 2014

Insulin-dependent diabetes mellitus is an autoimmune disease that is genetically linked to the HLA class II molecule DQ in humans and to MHC I-Ag7 in nonobese diabetic mice. The I-Ag7 β-chain is unique and contains multiple polymorphisms, at least one of which is shared with DQ alleles linked to insulin-dependent diabetes mellitus. This polymorphism occurs at position 57 in the β-chain, in which aspartic acid is mutated to a serine, a change that results in the loss of an interchain salt bridge between αArg76 and βAsp57 at the periphery of the peptide binding groove. Using mAbs we have identified alternative conformations of I-Ag7 class II molecules. By using an invariant chain construct with various peptides engineered into the class II-associated invariant chain peptide (CLIP) region we have found that formation of these conformations is dependent on the peptide occupying the binding groove. Blocking studies with these Abs indicate that these conformations are present at the cell surface and are capable of interactions with TCRs that result in T cell activation.
Enhanced Antiviral and Opsonic Activity of a Human Mannose-Binding Lectin and Surfactant Protein D Chimera
Authors: White Mitchell R. , Crouch Erika , Chang Donald , Sastry Kedarnath , Guo Ning , Engelich Georg , Takahashi Kazue , Ezekowitz R. Alan B. , Hartshorn Kevan L.
Published: April 2014

The carbohydrate recognition domains (CRDs) of human serum mannose-binding lectin (MBL) and pulmonary surfactant protein D (SP-D) have distinctive monosaccharide-binding properties, and their N-terminal and collagen domains have very different quaternary structures. We produced a chimeric protein containing the N terminus and collagen domain of human SP-D and the neck region and CRD of human MBL (SP-D/MBLneck+CRD) to create a novel human collectin. The chimera bound to influenza A virus (IAV), inhibited IAV hemagglutination activity and infectivity, and induced aggregation of viral particles to a much greater extent than MBL. Furthermore, SP-D/MBLneck+CRD caused much greater increases in neutrophil uptake of, and respiratory burst responses to, IAV than MBL. These results indicate that pathogen interactions mediated by the MBL CRD are strongly influenced by the N-terminal and collagen-domain backbone to which it is attached. The presence of the CRD of MBL in the chimera resulted in altered monosaccharide binding properties compared with SP-D. As a result, the chimera caused greater aggregation and neutralization of IAV than SP-D. Distinctive functional properties of collectin collagenous domains and CRDs can be exploited to generate novel human collectins with potential for therapy of influenza.
Colitis Induced by Enteric Bacterial Antigen-Specific CD4+ T Cells Requires CD40-CD40 Ligand Interactions for a Sustained Increase in Mucosal IL-12
Authors: Cong Yingzi , Weaver Casey T. , Lazenby Audrey , Elson Charles O.
Published: April 2014

C3H/HeJBir is a mouse substrain that is highly susceptible to colitis. Their CD4+ T cells react to Ags of the commensal enteric bacteria, and the latter can mediate colitis when activated by these Ags and transferred to histocompatible scid recipients. In this study, multiple long-term C3H/HeJBir CD4+ T cell (Bir) lines reactive to commensal enteric bacterial Ags have been generated. All these were Ag specific, pauciclonal, and Th1 predominant; most induced colitis uniformly after transfer to scid recipients. Lesions were focal and marked by increased expression of IL-12p40 and IFN-γ mRNA and protein. Pathogenic Bir T cell lines expressed CD40 ligand (CD40L) when cultured with Ag-pulsed APCs in vitro. Production of IL-12 was also increased in such cultures, an effect that was Ag- and T cell-dependent and required costimulation by CD40, but not by B7. The two Bir T cell lines that did not induce lesions after transfer failed to significantly express CD40L or increase IL-12 when cultured with Ag-pulsed APCs. Administration of anti-CD40L blocked disease expression induced by pathogenic T cells. We conclude that interactions in the colon mucosa between CD40L-expressing Bir Th1 cells with APCs endogenously loaded with commensal bacterial Ags are critical for sustained increases in local IL-12 production and progression to colitis.
Requirement of Hydrogen Peroxide Generation in TGF-β1 Signal Transduction in Human Lung Fibroblast Cells: Involvement of Hydrogen Peroxide and Ca2+ in TGF-β1-Induced IL-6 Expression
Authors: Junn Eunsung , Lee Kee Nyung , Ju Hyang Ran , Han Seung Hyun , Im Joo Young , Kang Hyung Sik , Lee Tae Ho , Bae Yun Soo , Ha Kwon Soo , Lee Zee Won , Rhee Sue Goo , Choi Inpyo
Published: April 2014

Stimulation of human lung fibroblast cells with TGF-β1 resulted in a transient burst of reactive oxygen species with maximal increase at 5 min after treatment. This reactive oxygen species increase was inhibited by the antioxidant, N-acetyl-l-cysteine (NAC). TGF-β1 treatment stimulated IL-6 gene expression and protein synthesis in human lung fibroblast cells. Antioxidants including NAC, glutathione, and catalase reduced TGF-β1-induced IL-6 gene expression, and direct H2O2 treatment induced IL-6 expression in a dose-dependent manner. NAC also reduced TGF-β1-induced AP-1 binding activity, which is involved in IL-6 gene expression. It has been reported that Ca2+ influx is stimulated by TGF-β1 treatment. EGTA suppressed TGF-β1- or H2O2-induced IL-6 expression, and ionomycin increased IL-6 expression, with simultaneously modulating AP-1 activity in the same pattern. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase/extracellular signal-related kinase kinase 1, suppressed TGF-β1- or H2O2-induced IL-6 and AP-1 activation. In addition, TGF-β1 or H2O2 increased MAPK activity which was reduced by EGTA and NAC, suggesting that MAPK is involved in TGF-β1-induced IL-6 expression. Taken together, these results indicate that TGF-β1 induces a transient increase of intracellular H2O2 production, which regulates downstream events such as Ca2+ influx, MAPK, and AP-1 activation and IL-6 gene expression.
IL-11 Selectively Inhibits Aeroallergen-Induced Pulmonary Eosinophilia and Th2 Cytokine Production
Authors: Wang Jingming , Homer Robert J. , Hong Lielie , Cohn Lauren , Lee Chun Guen , Jung Sungsoo , Elias Jack A.
Published: April 2014

IL-11 is a pleiotropic cytokine that induces tissue remodeling with subepithelial fibrosis when expressed in the airway. Its effects on the Th2-dominated airway inflammation that is characteristic of asthma, however, are poorly understood. To characterize the effects of IL-11 on Th2 tissue inflammation, we compared the inflammatory responses elicited by OVA in sensitized mice in which IL-11 is overexpressed in a lung-specific fashion (CC10-IL-11) with that in transgene− wild-type littermate controls. Transgene− and CC10-IL-11 transgene+ mice had comparable levels of circulating Ag-specific IgE after sensitization. OVA challenge of sensitized transgene− mice caused airway and parenchymal eosinophilic inflammation, Th2 cell accumulation, and mucus hypersecretion with mucus metaplasia. Exaggerated levels of immunoreactive endothelial cell VCAM-1, mucin (Muc) 5ac gene expression and bronchoalveolar lavage and lung IL-4, IL-5, and IL-13 protein and mRNA were also noted. In contrast, OVA challenge in CC10-IL-11 animals elicited impressively lower levels of tissue and bronchoalveolar lavage inflammation, eosinophilia, and Th2 cell accumulation, and significantly lower levels of VCAM-1 and IL-4, IL-5, and IL-13 mRNA and protein. IL-11 did not cause a comparable decrease in mucus hypersecretion, Muc 5ac gene expression, or the level of expression of RANTES, monocyte chemoattractant protein-2, or monocyte chemoattractant protein-3. In addition, IL-11 did not augment IFN-γ production demonstrating that the inhibitory effects of IL-11 were not due to a shift toward Th1 inflammation. These studies demonstrate that IL-11 selectively inhibits Ag-induced eosinophilia, Th2 inflammation, and VCAM-1 gene expression in pulmonary tissues.
Central Nervous System-Initiated Inflammation and Neurotrophism in Trauma: IL-1β Is Required for the Production of Ciliary Neurotrophic Factor
Authors: Herx Leonie M. , Rivest Serge , Yong V. Wee
Published: April 2014

Injury to the CNS results in the production and accumulation of inflammatory cytokines within this tissue. The origin and role of inflammation within the CNS remains controversial. In this paper we demonstrate that an acute trauma to the mouse brain results in the rapid elevation of IL-1β. This increase is detectable by 15 min after injury and significantly precedes the influx of leukocytes that occurs hours after. To confirm that IL-1β up-regulation is initiated by cells within the CNS, in situ hybridization for cytokine transcript was combined with cell type immunohistochemistry. The results reveal parenchymal microglia to be the sole source of IL-1β at 3 h postinjury. A role for CNS-initiated inflammation was addressed by examining the expression of the neurotrophic factor, ciliary neurotrophic factor (CNTF). Analysis of their temporal relationship suggests the up-regulation of CNTF by IL-1β, which was confirmed through three lines of evidence. First, the application of IL-1 receptor antagonist into the lesion site attenuated the up-regulation of CNTF. Second, the examination of corticectomized animals genetically deficient for IL-1β found no CNTF up-regulation. Third, the lack of CNTF elevation in IL-1β null mice was rescued through exogenous application of IL-1β into the lesion site. These findings provide the first evidence of the requirement for IL-1β in the production of CNTF following CNS trauma, and suggest that inflammation can have a beneficial impact on the regenerative capacity of the CNS.
B and T Cell Responses to the Spliceosomal Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in Normal and Lupus Mice
Authors: Dumortier Hélène , Monneaux Fanny , Jahn-Schmid Beatrice , Briand Jean-Paul , Skriner Karl , Cohen Phil L. , Smolen Joseph S. , Steiner Guenter , Muller Sylviane
Published: April 2014

Autoantibodies directed against spliceosomal heterogeneous nuclear ribonucleoproteins (hnRNPs) are a typical feature of rheumatoid arthritis, systemic lupus erythematosus, and mixed-connective tissue disease. With the aim of investigating a potential pathogenic role of these Abs, we have studied the Ab response to A2/B1 hnRNPs in different murine models of lupus. The specificity of anti-A2/B1 Abs was tested with a series of 14 overlapping synthetic peptides covering the region 1–206 of A2 that contains most of the epitopes recognized by patients’ Abs. A major epitope recognized very early during the course of the disease by Abs from most of MRL lpr/lpr mice but not from other lupus mice and from mice of different MHC haplotypes immunized against B1 was identified in residues 50–70. This peptide contains a highly conserved sequence RGFGFVTF also present in other hnRNPs and small nuclear ribonucleoproteins. Abs reacting with a second A2 epitope identified in residues 35–55 were detectable several weeks later, suggesting an intramolecular B cell epitope spreading during the course of the disease. We identified several T cell epitopes within the region 35–175 that generated an effective Th cell response with IL-2 and IFN-γ secretion in nonautoimmune CBA/J mice sharing the same MHC haplotype H-2k as MRL/lpr mice. None of the peptides stimulated T cells primed in vivo with B1. Because Abs to peptide 50–70 were detected significantly earlier than Abs reacting with other A2 peptides and the protein itself, it is possible that within the protein, this segment contains residues playing an initiator role in the induction of the anti-A2/B1 and antispliceosome Ab response.
Cutting Edge: T Cell-Specific Adapter Protein Inhibits T Cell Activation by Modulating Lck Activity
Authors: Sundvold Vibeke , Torgersen Knut Martin , Post Nicholas H. , Marti Francesc , King Philip D. , Røttingen John Arne , Spurkland Anne , Lea Tor
Published: April 2014

We previously reported the isolation of a cDNA encoding a T cell-specific adapter protein (TSAd). Its amino acid sequence contains an SH2 domain, tyrosines in protein binding motifs, and proline-rich regions. In this report we show that expression of TSAd is induced in normal peripheral blood T cells stimulated with anti-CD3 mAbs or anti-CD3 plus anti-CD28 mAbs. Overexpression of TSAd in Jurkat T cells interfered with TCR-mediated signaling by down-modulating anti-CD3/PMA-induced IL-2 promoter activity and anti-CD3 induced Ca2+ mobilization. The TCR-induced tyrosine phosphorylation of phospholipase C-γ1, SH2-domain-containing leukocyte-specific phosphoprotein of 76kDa, and linker for activation of T cells was also reduced. Furthermore, TSAd inhibited Zap-70 recruitment to the CD3ζ-chains in a dose-dependent manner. Consistent with this, Lck kinase activity was reduced 3- to 4-fold in COS-7 cells transfected with both TSAd and Lck, indicating a regulatory effect of TSAd on Lck. In conclusion, our data strongly suggest an inhibitory role for TSAd in proximal T cell activation.
Coordinate Regulation of the IL-4, IL-13, and IL-5 Cytokine Cluster in Th2 Clones Revealed by Allelic Expression Patterns
Authors: Kelly Ben L. , Locksley Richard M.
Published: April 2014

The cytokines IL-4, IL-13, and IL-5 are markers for the Th2 subset of effector T cells and are often expressed together. These cytokine genes are organized within 140 kb of orthologous DNA in both mouse and human. Using IL-4-expressing CD4+ T cell clones derived from F1 mice, we identified allelic polymorphisms for each of these cytokines and assessed the parental identity of the cytokine mRNAs. Both monoallelic and biallelic expression occurred for each gene and for an additional gene, IL-3, that lies with GM-CSF over 450 kb telomeric on the same chromosome. When coexpressed in T cell clones, IL-4 was expressed from the same allele as IL-13 or IL-5 in 81% of instances. In contrast, there was only 52% concordance of these three cytokines at the allelic level among clones that expressed IL-3. Independent expression of the cytokine alleles occurs commonly in T cells, but the clustered locus encompassing IL-4, IL-13, and IL-5 is subject to coordinate regulation.
A NK1.1+ Thymocyte-Derived TCR β-Chain Transgene Promotes Positive Selection of Thymic NK1.1+ αβ T Cells
Authors: Viret Christophe , Lantz Olivier , He Xin , Bendelac Albert , Janeway Charles A.
Published: April 2014

As a consequence of the peptide specificity of intrathymic positive selection, mice transgenic for a rearranged TCR β-chain derived from conventional αβ T lymphocytes frequently carry mature T cells with significant skewing in the repertoire of the companion α-chain. To assess the generality of such an influence, we generated transgenic (Tg) mice expressing a β-chain derived from nonclassical, NK1.1+ αβ T cells, the thymus-derived, CD1.1-specific DN32H6 T cell hybridoma. Results of the sequence analysis of genomic DNA from developing DN32H6 β Tg thymocytes revealed that the frequency of the parental α-chain sequence, in this instance the Vα14-Jα281 canonical α-chain, is specifically and in a CD1.1-dependent manner, increased in the postselection thymocyte population. In accordance, we found phenotypic and functional evidence for an increased frequency of thymic, but interestingly not peripheral, NK1.1+ αβ T cells in DN32H6 β Tg mice, possibly indicating a thymic determinant-dependent maintenance. Thus, in vivo expression of the rearranged TCR β-chain from a thymus-derived NK1.1+ Vα14+ T cell hybridoma promotes positive selection of thymic NK1.1+ αβ T cells. These observations indicate that the strong influence of productive β-chain rearrangements on the TCR sequence and specificity of developing thymocytes, which operates through positive selection on self-determinants, applies to both classical and nonclassical αβ T cells and therefore represents a general phenomenon in intrathymic αβ T lymphocyte development.
CD152 Ligation by CD80 on T Cells Is Required for the Induction of Unresponsiveness by Costimulation-Deficient Antigen Presentation
Authors: Chai Jian-Guo , Vendetti Silvia , Amofah Eunice , Dyson Julian , Lechler Robert
Published: April 2014

Two apparently contradictory observations have been made concerning peripheral T cell tolerance; costimulation-deficient Ag presentation leads to unresponsiveness, and CTLA4 (CD152) ligation is required for unresponsiveness to be induced. This issue was addressed using a CD80− CD86low B cell line to present Ag to DO.11.10 naive CD4+ T cells. Proliferation was substantially enhanced by anti-CD80 or anti-CD152, but was inhibited by anti-CD86. Furthermore, anti-CD80 partially, and anti-CD152 totally protected cloned DO.11.10 T cells from the induction of unresponsiveness following culture with peptide and Chinese hamster ovary H2-Ad+ CD80− CD86− cells. Fab of anti-CD80 caused similar enhancement, and coimmobilized anti-CD80 failed to costimulate the anti-CD3 response of purified T cells, indicating that direct signaling by anti-CD80 was not responsible for these effects. The possibility that anti-CD80 liberated CD28 molecules that were sequestered by the T cell-expressed CD80, enabling them to coaggregate with TCR:CD3 complexes was excluded by finding that anti-CD80 and anti-CD152 individually caused maximal enhancement, rather than having additive effects. These data suggest that T cell-expressed CD80 has a regulatory function and plays a key role in the induction of unresponsiveness due to costimulation-deficient Ag presentation by the ligation of CD152 on neighboring, or even the same, T cell.
Either B7 Costimulation or IL-2 Can Elicit Generation of Primary Alloreactive CTL
Authors: McAdam Alexander J. , Gewurz Benjamin E. , Farkash Evan A. , Sharpe Arlene H.
Published: April 2014

B7-1 and B7-2 are important costimulatory molecules in the activation of T cell immunity. We have used mice made genetically deficient in either or both B7 molecules to determine the role of B7 molecules in activation of primary alloreactive CTL. The absence of either B7-1 or B7-2 did not alter generation of CTL from unfractionated lymphocytes, but the absence of B7-2 greatly decreased CTL generation from purified CD8+ responder cells. However, if B7-1 was induced on the stimulating cells then CTL generation was restored to wild-type levels. Absence of both B7-1 and B7-2 from MLR using whole splenocytes resulted in a profound reduction in generation of CTL. This could completely be reversed by the addition of IL-2. B7 molecules could directly costimulate CD8+ cells, as purified CD8+ cells developed into mature CTL when stimulated with wild-type APC, but not with B7-deficient APC. Again, IL-2 could drive CTL generation from purified CD8+ cells, even in the absence of B7 molecules. Taken together, these results demonstrate an important role for B7 costimulation in CTL generation.
IL-18 Directs Autoreactive T Cells and Promotes Autodestruction in the Central Nervous System Via Induction of IFN-γ by NK Cells
Authors: Shi Fu-Dong , Takeda Kiyoshi , Akira Shizuo , Sarvetnick Nora , Ljunggren Hans-Gustaf
Published: April 2014

IL-18 promotes NK cell and Th1 cell activity and may bridge innate and adaptive immune responses. Myelin oligodendrocyte glycoprotein (MOG) is a myelin component of the CNS and is a candidate autoantigen in multiple sclerosis. In the present study we show that IL-18-deficient (IL-18−/−) mice are defective in mounting autoreactive Th1 and autoantibody responses and are resistant to MOG35–55 peptide-induced autoimmune encephalomyelitis. IL-18 administration enhances the disease severity in wild-type mice and restores the ability to generate Th1 response in the IL-18−/− mice. This restoration was abrogated in NK cell-depleted mice, indicating that the action of IL-18 in promoting the generation of MOG-specific Th cells was dependent on NK cells. Furthermore, transfer of NK cells from recombinase-activating gene 1−/− mice, but not from recombinase-activating gene 1/IFN-γ−/− mice, rescued the defective Th1 responses in IL-18−/− mice and rendered IL-18−/− mice susceptible to the induction of autoimmune encephalomyelitis. Thus, IL-18 can direct autoreactive T cells and promote autodestruction in the CNS at least in part via induction of IFN-γ by NK cells.
An Intra-Peyer’s Patch Gene Transfer Model for Studying Mucosal Tolerance: Distinct Roles of B7 and IL-12 in Mucosal T Cell Tolerance
Authors: Chen Yiguang , Song Kaimei , Eck Stephen L. , Chen Youhai
Published: April 2014

Development of mucosal immunity and tolerance requires coordinated expression of a number of genes within the mucosa-associated lymphoid tissue (MALT). To study the roles of these genes in the MALT, we have established a MALT-specific gene transfer model using replication-defective adenovirus as vector. In this model, the target gene of interest is directly delivered into the Peyer’s patch by intra-Peyer’s patch injection of the recombinant virus. Using this gene transfer model, we investigated the roles of B7-1 and IL-12 in the development of mucosal tolerance. We found that intra-Peyer’s patch injection of OVA induced Ag-specific T cell hyporesponsiveness, as manifested by decreased T cell proliferation and IL-2/IFN-γ production upon subsequent immune challenge. Intra-Peyer’s patch B7-1 gene transfer at the time of OVA administration partially reversed the inhibition of T cell proliferation and IL-2 secretion, but had no effect on IFN-γ production. By contrast, intra-Peyer’s patch IL-12 gene transfer completely restored T cell proliferation and IFN-γ secretion and partially reversed IL-2 inhibition. Using an adoptive TCR transgenic model, we further demonstrated that B7 and IL-12 played distinct roles during the inductive phase of mucosal tolerance. B7 selectively increased T cell proliferation and IL-2 secretion without affecting IFN-γ production, whereas IL-12 increased both IL-2 and IFN-γ production. These results indicate that B7 alone may not be sufficient to abrogate mucosal tolerance, and that cytokines such as IL-12 may also be required. Based on these findings, we propose a new model to explain the paradoxical roles of B7 in mucosal immunity and tolerance.
Exposure to Ultraviolet Radiation Causes Dendritic Cells/Macrophages to Secrete Immune-Suppressive IL-12p40 Homodimers
Authors: Schmitt David A. , Ullrich Stephen E.
Published: April 2014

UV-induced immune suppression is a risk factor for sunlight-induced skin cancer. Exposure to UV radiation has been shown to suppress the rejection of highly antigenic UV-induced skin cancers, suppresses delayed and contact hypersensitivity, and depress the ability of dendritic cells to present Ag to T cells. One consequence of UV exposure is altered activation of T cell subsets. APCs from UV-irradiated mice fail to present Ag to Th1 T cells; however, Ag presentation to Th2 T cells is normal. While this has been known for some time, the mechanism behind the preferential suppression of Th1 cell activation has yet to be explained. We tested the hypothesis that this selective impairment of APC function results from altered cytokine production. We found that dendritic cells/macrophages (DC/Mφ) from UV-irradiated mice failed to secrete biologically active IL-12 following in vitro stimulation with LPS. Instead, DC/Mφ isolated from the lymphoid organs of UV-irradiated mice secreted IL-12p40 homodimer, a natural antagonist of biologically active IL-12. Furthermore, when culture supernatants from UV-derived DC/Mφ were added to IL-12-activated T cells, IFN-γ secretion was totally suppressed, indicating that the IL-12p40 homodimer found in the supernatant fluid was biologically active. We suggest that by suppressing DC/Mφ IL-12p70 secretion while promoting IL-12p40 homodimer secretion, UV exposure preferentially suppress the activation of Th1 cells, thereby suppressing Th-1 cell-driven inflammatory immune reactions.
Subset-Specific Regulation of the Lymphatic Exit of Recirculating Lymphocytes In Vivo
Authors: Young Alan J. , Marston Wendy L. , Dudler Lisbeth
Published: April 2014

The blood-to-lymph recirculation of lymphocytes is required for the maintenance of immune surveillance and the dissemination of memory. Although the ability of lymph-borne cells to recirculate has been well documented, relatively less is known about the migration capacity of PBLs. We have found a clear preference for PBLs to recirculate through s.c. rather than intestinal lymph nodes. This preference could be directly attributed to the migratory characteristics of γδ-T cells. γδ-T cells were found to express significantly higher levels of L-selectin than other subsets, suggesting that at least some of this preferential migration could be attributed to their interaction with ligands on vascular endothelium. More detailed experiments showed that γδ-T cells migrated through lymph nodes with greater efficiency than αβ T cells or B cells, which clearly indicated an enhanced ability of γδ-T cells to exit lymph nodes in the efferent lymph independent of entry from the blood. This hypothesis was supported by histological examination, where γδ-T cells were found almost exclusively in the interfollicular traffic areas within lymph nodes. These data indicate that γδ-T cells are the most active recirculating lymphocyte subset in ruminants and suggest new mechanisms to regulate the traffic of lymphocyte subsets through normal lymph nodes.
Nonstandard Peptide Binding Revealed by Crystal Structures of HLA-B*5101 Complexed with HIV Immunodominant Epitopes
Authors: Maenaka Katsumi , Maenaka Taeko , Tomiyama Hiroko , Takiguchi Masafumi , Stuart David I. , Jones E. Yvonne
Published: April 2014

The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography. In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171. This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the α1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.
Pepsin-Mediated Processing of the Cytoplasmic Histone H2A to Strong Antimicrobial Peptide Buforin I
Authors: Kim Hun Sik , Yoon Ho , Minn Il , Park Chan Bae , Lee Won Taek , Zasloff Michael , Kim Sun Chang
Published: April 2014

The intestinal epithelium forms a first line of innate host defense by secretion of proteins with antimicrobial activity against microbial infection. Despite the extensive studies on the antimicrobial host defense in many gastrointestinal tracts, little is known about the antimicrobial defense system of the stomach. The potent antimicrobial peptide buforin I, consisting of 39 aa, was isolated recently from the stomach tissue of an Asian toad, Bufo bufo gargarizans. In this study we examined the mechanism of buforin I production in toad stomach tissue. Buforin I is produced by the action of pepsin isozymes, named pepsin Ca and Cb, cleaving the Tyr39-Ala40 bond of histone H2A. Immunohistochemical analysis revealed that buforin I is present extracellularly on the mucosal surface, and unacetylated histone H2A, a precursor of buforin I, is localized in the cytoplasm of gastric gland cells. Furthermore, Western blot analysis showed that buforin I is also present in the gastric fluids, and immunoelectron microscopy detected localization of the unacetylated histone H2A in the cytoplasmic granules of gastric gland cells. The distinct subcellular distribution of the unacetylated histone H2A and the detection of the unacetylated buforin I both on the mucosal surface and in the lumen suggest that buforin I is produced from the cytoplasmic unacetylated histone H2A secreted into the gastric lumen and subsequently processed by pepsins. Our results indicate that buforin I along with pepsins in the vertebrate stomach may contribute to the innate host defense of the stomach against invading microorganisms.
Dual Role of the HIV-1 Vpr Protein in the Modulation of the Apoptotic Response of T Cells
Authors: Conti Lucia , Matarrese Paola , Varano Barbara , Gauzzi Maria Cristina , Sato Akihiko , Malorni Walter , Belardelli Filippo , Gessani Sandra
Published: April 2014

We investigated the effect of vpr, physiologically expressed during the course of an acute HIV-1 infection, on the response of infected cells to apoptotic stimuli as well as on the HIV-induced apoptosis. At 48 h after infection, Jurkat cells exhibited a lower susceptibility to undergo apoptosis with respect to uninfected cells. This effect was not observed following infection with either a vpr-mutated virus or a wild-type strain in the presence of antisense oligodeoxynucleotides targeted at vpr mRNA. Single-cell analysis, aimed at simultaneously identifying apoptotic and infected cells, revealed that resistance to apoptosis correlated with productive infection. Notably, vpr-dependent protection from induced apoptosis was also observed in HIV-1-infected PBMC. In contrast, at later stages of infection, a marked increase in the number of cells spontaneously undergoing apoptosis was detected in infected cultures. This virus-induced apoptosis involved vpr expression and predominantly occurred in productively infected cells. These results indicate that HIV-1 vpr can exert opposite roles in the regulation of apoptosis, which may depend on the level of its intracellular expression at different stages of HIV-1 infection. The dual function of vpr represents a novel mechanism in the complex strategy evolved by HIV to influence the turnover of T lymphocytes leading to either viral persistence or virus release and spreading.
The B Subunit of Shiga Toxin Fused to a Tumor Antigen Elicits CTL and Targets Dendritic Cells to Allow MHC Class I-Restricted Presentation of Peptides Derived from Exogenous Antigens
Authors: Haicheur Nacilla , Bismuth Emmanuelle , Bosset Sophie , Adotevi Olivier , Warnier Guy , Lacabanne Valérie , Regnault Armelle , Desaymard Catherine , Amigorena Sebastian , Ricciardi-Castagnoli Paola , Goud Bruno , Fridman Wolf H. , Johannes Ludger , Tartour Eric
Published: April 2014

Immunization with peptide or recombinant proteins generally fails to elicit CTL, which are thought to play a key role in the control of virus-infected cells and tumor growth. In this study we show that the nontoxic B subunit of Shiga toxin fused to a tumor peptide derived from the mouse mastocytoma P815 can induce specific CTL in mice without the use of adjuvant. The Shiga B subunit acts as a vector rather than as an adjuvant, because coinjection of the tumor peptide and the B subunit as separate entities does not lead to CTL induction. We also demonstrated that in vitro the B subunit mediates the delivery of various exogenous CD8 T cell epitopes into the conventional MHC class I-restricted pathway, as this process is inhibited by brefeldin A and lactacystin and requires a functional TAP system. In contrast to other nonviral methods for transport of exogenous Ags into the endogenous MHC class I pathway that involve macropinocytosis or phagocytosis, the Shiga B subunit targets this pathway in a receptor-dependent manner, namely via binding to the glycolipid Gb3. Because this receptor is highly expressed on various dendritic cells, it should allow preferential targeting of the Shiga B subunit to these professional APCs. Therefore, the Shiga B subunit appears to represent an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL without the need for adjuvant.
Nitric Oxide Is Neither Necessary Nor Sufficient for Resolution ofPlasmodium chabaudiMalaria in Mice
Authors: van der Heyde Henri C. , Gu Yang , Zhang Qiang , Sun Guang , Grisham Matthew B.
Published: April 2014

Malaria is a life-threatening re-emerging disease, yet it is still not clear how bloodstage malarial parasites are killed. Nitric oxide (NO), which has potent anti-microbial activity, may represent an important killing mechanism. The production of NO during descending Plasmodium chabaudi parasitemia, a period when parasites are killed by the immune response, supports this concept. However, NOS20/0 and NOS30/0 mice as well as mice treated with NO synthase 2 (NOS2) inhibitors do not develop exacerbated malaria, indicating that NO production is not necessary for the suppression of P. chabaudi parasitemia. It is possible due to the plasticity in the immune response during malaria that Ab-mediated immunity is enhanced in the absence of NO, thereby explaining the lack of exacerbated malaria in NOS-deficient mice even though NO may function in protection. However, NOS2- and B cell-deficient mice, which cannot use Ab-mediated immunity, suppress their parasitemia with a similar time course as B cell-deficient controls. C57BL/6 mice treated with Propionibacterium acnes to elicit high levels of macrophage-derived NO have a similar time course of P. chabaudi parasitemia as P. acnes-treated NOS20/0 mice, which do not produce NO; this indicates that NO is not sufficient for parasite killing. Collectively, these results indicate that NO is not necessary or sufficient to resolve P. chabaudi malaria.
Autoantibody Responses and Pathology Regulated by B7-1 and B7-2 Costimulation in MRL/lprLupus
Authors: Liang Bailin , Kashgarian Michael J. , Sharpe Arlene H. , Mamula Mark J.
Published: April 2014

The activation of T lymphocytes requires both Ag-mediated signaling through the TCR as well as costimulatory signals transmitted through B7-1 and/or B7-2 with CD28. The interference of B7-mediated costimulatory signals has been proposed as one immunotherapeutic intervention for the prevention autoimmune disease. This study has examined autoantibody responses and autoimmune pathology in a murine model of human systemic lupus erythematosus (SLE), the MRL-lpr/lpr mouse, genetically deficient in B7-1 or B7-2, or in mice treated with B7-1/B7-2 blocking Abs. In contrast to other studies of murine models of SLE, MRL-lpr/lpr mice treated with B7 blocking Abs exhibit strong anti-small nuclear ribonucleoprotein (snRNP) and anti-DNA autoantibody responses with some changes in isotype switching as compared with untreated animals. All MRL-lpr/lpr mice deficient in B7-1 or B7-2 produce anti-snRNP and anti-DNA titers with isotypes virtually identical with wild-type animals. However, the absence of B7-2 costimulation did interfere with the spontaneous activation and the accumulation of memory CD4+ or CD8+ T lymphocytes characteristic of wild-type MRL-lpr/lpr mice. IgG and C3 complement deposition was less pronounced in the kidneys of B7-2 deficient MRL-lpr/lpr mice, reflecting their lessor degree of glomerulonephritis. By comparison, B7-1-deficient MRL-lpr/lpr mice had more severe IgG and C3 deposits in glomeruli.