bluuebunny/crossref_metadata_2025 huggingface dataset:
The CD8β polypeptide is required for the recognition of an altered peptide ligand as an agonist
Authors: Renard Valéry , Delon Jérôme , Luescher Immanuel F. , Malissen Bernard , Vivier Eric , Trautmann Alain
Published: March 2007

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin‐2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 α and β chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen‐presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8β in the CD8 co‐receptor dimers expressed at the T cell surface.
A novel SP‐1 site in the human interleukin‐1β promoter confers preferential transcriptional activity in keratinocytes
Authors: Husmann Matthias , Jehnichen Petra , Jahn Bernhard , Schlosshan Dominik , Romahn Eric , Marx Jürgen
Published: March 2007

To investigate the mechanisms of transcriptional activation of interleukin‐1β (IL‐1β) in non‐monocytic cells, we constructed a series of reporter plasmids with the bacterial chloramphenicol acetyltransferase gene linked to various parts of the human IL‐1β promoter and performed transient transfection experiments. We identified a promoter segment that activates transcription most efficiently in keratinocytes. Electrophoretic mobility shift assays (EMSA) with a 43‐mer oligonucleotide derived from the functionally identified cis‐acting element revealed specific complexes. By competition analysis with transcription factor consensus sequence oligonucleotides and by immunosupershift, transcription factor SP‐1 or a closely related protein was shown to bind to this regulatory element. The closest match to the known SP‐1 consensus sequence within the respective region is a TCCCCTCCCCT motif. Mutation of this motif almost completely, and specifically, abolished the binding of two low‐mobility complexes and led to a 95% decrease of constitutive transcriptional activation of a reporter construct IL‐1β (− 170/+ 108). Likewise, activation of this reporter construct by tumor necrosis factor‐α depended on the SP‐1 site. These observations suggest that a so‐far‐unrecognized SP‐1 site in the human IL‐1β promoter may participate in the transcriptional regulation of this gene in keratinocytes.
Cytotoxic reactivity of gut lamina propria CD4+ αβ T cells in SCID mice with colitis
Authors: Bonhagen Kerstin , Thoma Stefan , Bland Paul , Bregenholt Søren , Rudolphi Angelika , Claesson Mogens H. , Reimann Jörg
Published: March 2007

Polyclonal, mucosa‐seeking memory/effector CD4+ T cells containing a large fraction of blasts activated in situ accumulate in the gut lamina propria of severe‐combined immunodeficient (SCID) mice developing colitis after CD4+ T cell transplantation. CD4+ T cells isolated from different repopulated lymphoid tissues of transplanted SCID mice proliferate in vitro in the presence of interleukin (IL)‐2 + IL‐7. CD3 ligation enhances this cytokine‐supported proliferation in CD4+ T cells from the spleen and the mesenteric lymph node of transplanted SCID mice; CD3 ligation suppresses the cytokine‐supported proliferation in CD4+ T cells from the gut lamina propria in a cell density‐ and dose‐dependent manner. Almost all CD4+ T cells from repopulated lymphoid tissues of transplanted SCID mice express CD95 (Fas) on the cell surface, and a large fraction of CD4+ T cells from the gut lamina propria of transplanted SCID mice express the Fas ligand on the surface. Gut lamina propria CD4+ T cells show Fas‐dependent cytotoxicity. A large fraction of gut lamina propria CD4+ T cells that infiltrate the inflamed colon in transplanted SCID mice are activated in situ and many CD4+ T cells are apoptotic. Hence, a large fraction of colitis‐inducing CD4+ T cells undergo activation‐induced cell death in situ and can damage other cells through Fas‐dependent cytotoxicity.
Peripheral tolerance of CD4 T cells following local activation in adolescent mice
Authors: Förster Irmgard , Lieberam Ivo
Published: March 2007

In addition to thymic T cell selection, post‐thymic mechanisms of tolerance induction are required to eliminate autoreactive T cells with specificities for peripheral self antigens. While CD8+ T cells can recognize their target antigen on a wide variety of cell types, CD4+ T cells generally depend on the presence of specialized antigen‐presenting cells. Because of this fundamental difference in antigen recognition peripheral tolerance of CD4+ T cells appears more difficult to achieve than of CD8+ T cells. Utilizing T cell receptor (TCR)‐transgenic mice in which CD4+ T cells specific for a pancreatic β cell neoantigen (the simian virus 40 T antigen) are constantly generated at low frequency, we have now established a mouse model of peripheral, tissue‐specific CD4+ T cell tolerance. In these animals, tolerance is preceded by a phase of activation of the autoreactive T cells as characterized by up‐regulation of CD69 and CD44, and down‐regulation of the L‐selectin lymph node homing receptor. T antigen‐specific T cells bearing this phenotype can be detected in the local lymphoid environment of the pancreas but not in more remote locations like axillary or inguinal lymph nodes. The proportion of activated, autoreactive T cells is maximal at 2–3 weeks of age, after which these cells are gradually deleted from the peripheral lymphocyte pool. We further demonstrate that deletion of the autoreactive T cells does not occur in TCR‐tansgenic mice bred to the RAG‐1‐deficient background in which the transgenic T cells represent the only functional lymphocyte population.
T cell receptor α gene rearrangement and transcription in adult thymic γδ cells
Authors: Mertsching Elisabeth , Wilson Anne , MacDonald H. Robson , Ceredig Rhodri
Published: March 2007

T cells belong to two separate lineages based on surface expression of αβ or γδ T cell receptors (TCR). Since during thymus development TCR β, γ, and δ genes rearrange before α genes, and γδ cells appear earlier than αβ cells, it has been assumed that αδ cells are devoid of TCR α rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic γδ cells undergo VJα rearrangements more frequently than immature αβ lineage thymic precursors. Sequence analysis shows VJα rearrangements in γδ cells to be mostly (70 %) nonproductive. Furthermore, VJα rearrangements in γδ cells are transcribed normally and, as shown by analysis of TCR β‐/‐ mice, occur independently of productive VDJβ rearrangements. These data are interpreted in the context of a model in which precursors of αβ and γδ cells differ in their ability to express a functional pre‐TCR complex.
The intensity of T cell receptor engagement determines the cytokine pattern of human allergen‐specific T helper cells
Authors: Carballido José M. , Faith Alexander , Carballido‐Perrig Nicole , Blaser Kurt
Published: March 2007

Enhanced production of T helper (Th)2 cytokines by allergen‐specific Th cells plays a major role in the induction and maintenance of IgE‐mediated allergic disorders. The mechanism that triggers this type of response in atopic individuals is not fully understood. Allergen‐specific human Th cell clones produce interleu‐kin (IL)‐4 and low or undetectable levels of interferon (IFN)‐γ after stimulation with low concentrations of antigen. However, these Th cell clones are capable of generating significant amounts of IFN‐γ after optimal activation through their T cell receptor (TcR). Allergen‐specific Th cell clones isolated from allergic individuals required higher doses of antigen to reach the plateau of proliferation and to generate Th0 cytokine responses than their counterparts isolated from nonal‐lergic subjects. On the other hand, if allergen was replaced by anti‐CD3 monoclonal antibody (mAb), both allergic and nonallergic Th cell clones attained the highest level of proliferation and significant IFN‐γ production in response to equivalent concentrations of anti‐CD3 mAb. These results indicate that the strength of T cell ligation, which can be modulated by the availability of the TcR ligand, controls the balance of Th1/Th2 cytokines produced by memory Th cells in vitro. In the particular case of bee venom phospholipase A2, it is shown that the expression of allergen‐specific surface Ig on antigen‐presenting B cells has little influence on antigen uptake and therefore in determining the levels of T cell activation and cytokine production. Alternatively, the affinity of particular major histocompatibility complex class II molecules on antigen‐presenting cells for allergen‐derived peptides might determine the amount of specific ligand presented to the Th cells and play a decisive role skewing the Th cell cytokine production towards Th1 or Th2 phenotypes. These findings, which are consistent with the changes in cytokine patterns observed following clinical hyposensitization, suggest that polarized human Th2 cell subsets still retain the capacity to modulate their cytokine pattern.
Immune modulation with interleukin‐4 and interleukin‐10 prevents crescent formation and glomerular injury in experimental glomerulonephritis
Authors: Tipping Peter G. , Kitching A. Richard , Huang Xiao Ru , Mutch David A. , Holdsworth Stephen R.
Published: March 2007

Crescentic glomerulonephritis (GN) demonstrates immunopathological features of a T helper (Th)1‐directed delayed‐type hypersensitivity (DTH) response. The capacity of Th2 cytokines to attenuate crescentic glomerular injury in this disease was examined by administering interleukin (IL)‐4 and IL‐10, singly and in combination. GN was induced by i.v. administration of sheep anti‐mouse glomerular basement membrane (GBM) globulin to mice sensitized to sheep globulin 10 days earlier. Treatment (2.5 μg, i.p.) with IL‐4, IL‐10, or both IL‐4 and IL‐10 (IL‐4+10), was started 1 h before sensitization and continued daily until the end of the study (10 days after administration of anti‐GBM globulin). Control mice treated with PBS developed GN with glomerular accumulation of T cells and macrophages, crescents in 42.5 ± 4.5 % of glomeruli (normal 0 %), proteinuria (8.3 ± 0.9 mg/24 h, normal 0.74 ± 0.08 mg/24 h, p < 0.001) and renal impairment (creatinine clearance [cr/cl]: 93 ± 12 μ1/min, normal 193 ± 10 μ1/min, p<0.001). 1reatment with either IL‐4, IL‐10, or IL‐4+10 prevented crescent formation (crescentic glomeruli: 0.8 ± 0.5, 1.2 ± 0.9, and 1.4 ± 1.0 %, respectively, all p<0.01 compared to control) and attenuated proteinuria (3.6 ± 1.0, 2.2 ± 0.5, and 2.9 ± 0.5 mg/24 h, respectively, all p<0.01 compared to control). IL‐4+10 prevented development of renal impairment (cr/cl: 183 ± 22 μ1/min); IL‐10 given alone limited the decline in renal function (cr/cl: 150 ± 20 μ1/min), but IL‐4 alone did not provide any significant protection (cr/cl: 121 ± 17 μ1/min). All treatments markedly diminished glomerular T cell and macrophage accumulation, reduced interferon‐γ production by splenic T cells, prevented cutaneous DTH to the disease‐initiating antigen and reduced antigen‐specific immunoglobulin of the IgG2a and IgG3 isotypes. These data demonstrate that crescentic GN and renal impairment can be prevented by administration of Th2 cytokines and that this effect is associated with attenuation of the Th1 response to the disease‐initiating antigen.
Cleavage of the low‐affinity receptor for human IgE (CD23) by a mite cysteine protease: Nature of the cleaved fragment in relation to the structure and function of CD23
Authors: Schulz Oliver , Sutton Brian J. , Beavil Rebecca L. , Shi Jianguo , Sewell Herb F. , Gould Hannah J. , Laing Peter , Shakib Farouk
Published: March 2007

Der p I, a cysteine protease representing a major allergen of the house dust mite Dermatophagoides pteronyssinus, has recently been shown to cleave CD23 from the surface of cultured human B cells (RPMI 8866 B cell line). We have now undertaken a detailed investigation of CD23 cleavage by Der p I. We demonstrate that Der p I cleaves CD23 at two sites (Ser155‐Ser156 and Glu298‐Ser299) to produce a 17‐kDa fragment containing the lectin domain and only part of the C‐terminal tail. No such effect was demonstrable with mouse CD23, a finding which was anticipated based on its lack of the cleavage sites identified on human CD23. Based on the cleavage pattern and the model of CD23, we propose a sequence of events leading to the liberation of the 17‐kDa soluble CD23 fragment. The biological significance of such cleavage is underlined by the demonstration that Der p I‐treated B lymphocytes lose their ability to bind IgE, and that the 17‐kDa fragment (amino acids 156–298) contains the minimum structural requirement (amino acids 156–288) for binding to both IgE and CD21.
Antigen‐presenting cells pulsed with unfractionated tumor‐derived peptides are potent tumor vaccines
Authors: Nair Smita K. , Boczkowski David , Snyder David , Gilboa Eli
Published: March 2007

Vaccination with peptides isolated from tumor cells circumvents the need for identifying specific tumor rejection antigens and extends the use of active immunotherapy to the majority of cancers where specific tumor antigens have not yet been identified. In this study, we examined the efficacy of tumor vaccines composed of unfractionated tumor peptides presented by antigen‐presenting cells (APC) to induce cytotoxic T lymphocyte (CTL) responses and tumor immunity. RMA‐S cells pulsed with peptides isolated from ovalbumin (OVA)‐expressing tumor cells were highly effective at inducing primary, OVA‐specific CTL responses in vitro and priming CTL responses in vivo. In addition, tumor peptide‐pulsed RMA‐S cells induced protective immunity in mice when challenged with OVA‐expressing tumor cells. To enhance the clinical relevance of these studies, cells pulsed with tumor peptides were evaluated in the poorly immunogenic, B16/F10.9 melanoma post‐surgical metastasis model. Treatment of tumor‐bearing mice with peptide‐pulsed RMA‐S cells or with adherent splenocytes (enriched for professional APC) caused a significant reduction in lung metastases. The antimetastatic effect of peptide‐pulsed splenocytes could be further enhanced by pretreating the cells with antisense oligonucleotides directed against the TAP‐2 gene which was previously shown to increase the density of specific peptide/MHC class I complexes and thereby improve the APC function of the treated cells (Nair et el., J. Immunol. 1996. 156: 1772). This study suggests that APC loaded with unfractionated peptides derived from poorly immunogenic, highly metastatic tumor cells may represent a potent form of tumor vaccine.
Cloning of novel immunoglobulin superfamily receptors expressed on human myeloid and lymphoid cells: Structural evidence for new stimulatory and inhibitory pathways
Authors: Samaridis Jacqueline , Colonna Marco
Published: March 2007

We have identified two novel human cDNA encoding transmembrane proteins of the immunoglobulin superfamily (IgSF). The two cDNA, called immunoglobulin‐like transcripts 1 and 2 (ILT1 and ILT2), are expressed in myeloid and lymphoid cells and are homologous to bovine Fcγ2R, human killer cell inhibitory receptors (KIR), human FcαR, and mouse gp49. Furthermore, ILT1 and ILT2 are encoded on chromosome 19, as are FcαR and KIR. While the ILT1 and ILT2 extracellular domains are homologous, the transmembrane and cytoplasmic domains differ substantially. ILT1 has an arginine within the transmembrane region, followed by a short cytoplasmic tail, similar to human FcαRI and bovine Fcγ2R. ILT2 has a long cytoplasmic tail, which contains two YxxV and two YxxL pairs similar to the immunoreceptor tyrosine‐based inhibitory motifs in KIR that are known to bind the phosphotyrosine phosphatase SHP‐1. These cytoplasmic features suggest that ILT1 and ILT2 may mediate novel transmembrane signals by which myeloid and lymphoid cell responses can be either activated or inhibited.
Interferon‐γ is essential for the development of cerebral malaria
Authors: Rudin Werner , Favre Nicolas , Bordmann Gérard , Ryffel Bernhard
Published: March 2007

Infection with Plasmodium berghei ANKA (PbA) causes fatal cerebral malaria (CM). While a pathogenic role for tumor necrosis factor (TNF) has been established, we asked whether a disruption of interferon‐γ (IFN‐γ) signaling would modulate CM. We demonstrate here that IFN‐γR‐deficient mice are completely protected from CM. PbA‐induced release of TNF and up‐regulation of endothelial intercellular adhesion molecule (ICAM)‐1 expression, recruitment of mononuclear cells, and cerebral microvascular damage with vascular leakage occur only in wild‐type mice. Protected mice die at a later time of severe anemia and overwhelming parasitemia. Resistance to CM in IFN‐γR‐deficient mice is associated with reduced serum TNF levels, reduced interleukin‐12 expression in the brain and increased T‐helper 2 cytokines. In conclusion, IFN‐γ is apparently required for PbA‐induced endothelial ICAM‐1 up‐regulation and subsequent microvascular pathology, resulting in fatal CM. In the absence of IFN‐γ signaling, ICAM‐1 and TNF up‐regulation is reduced; hence, PbA infection fails to cause fatal CM.
Selective down‐regulation of Th2 immune responses following treatment with antigen‐coupled splenocytes
Authors: Soldera Sylvelie , McSorley Stephen J. , Glaichenhaus Nicolas
Published: March 2007

Intravenous injection of antigen‐coupled splenocytes has been widely used to induce specific tolerance to a variety of antigens. In this study, we investigated the effects of such a treatment on Th1 and Th2 antigen‐specific immune responses. Using both well‐characterized model antigens and crude homogenates from Leishmania major promastigotes, we found that intravenous injection of antigen‐coupled splenocytes strongly down‐regulated antigen‐specific Th2 responses but had no or only moderate effects on Th1 responses. Because the susceptibility of inbred strains of mice to murine leishmaniasis has been found to be correlated with a strong Th2 response against parasite antigens, we investigated whether administration of splenocytes chemically coupled to parasite antigens could protect susceptible mice from murine leishmaniasis. We found that this was indeed the case and further demonstrated that protection was associated with a strong decrease in the number of parasite‐specific Th2‐like cells. Because administration of antigen‐coupled splenocytes is believed to induce ligation of the T cell receptor complex without inducing a co‐stimulatory signal, our results further suggest that priming of Th1 cells is less dependent on co‐stimulatory signals than the priming of Th2 cells.
Increased severity of experimental autoimmune encephalomyelitis, chronic macrophage/microglial reactivity, and demyelination in transgenic mice producing tumor necrosis factor‐α in the central nervous system
Authors: Taupin Véronique , Renno Toufic , Bourbonnière Lyne , Peterson Alan C. , Rodriguez Moses , Owens Trevor
Published: March 2007

Tumor necrosis factor‐α (TNF‐α) is an inflammatory cytokine implicated in a number of autoimmune diseases. Apoptotic cell death is induced by TNF‐α in vitro, and has been suggested as one cause of autoimmune pathology, including autoimmune demyelinating diseases where oligodendrocytes are a target of immune attack. TNF‐α also regulates macrophage activity which could contribute to autoimmune inflammation. We have expressed TNF‐α at disease‐equivalent levels in the central nervous system of transgenic mice, using a myelin basic protein (MBP) promoter. These mice were normal and showed no spontaneous pathology, but they developed experimental autoimmune encephalomyelitis (EAE) with greater severity than nontransgenic controls when immunized with MBP in adjuvant. Unlike nontransgenic controls, EAE then progressed to a nonabating demyelinating disease. Macrophage/microglial reactivity was evident in demyelinating lesions in spinal cord, but T cells were not detected during chronic disease. The participation of TNF‐α in the demyelinating process is thus more probably due to the perpetuation of macrophage/microglial activation than to direct cytotoxicity of myelin or oligodendroglia.
Chemoattractant receptors for interleukin‐8 and C5a: expression on peripheral blood leukocytes and differential regulation on HL‐60 and AML‐193 cells by vitamin D3 and all‐trans rètinoic acid
Authors: Zahn Stefan , Zwirner JÖRg , Spengler Hans‐Peter , Götze Otto
Published: March 2007

Two homologous high‐affinity receptors for the chemoattractant interleukin‐8, IL‐8RA and IL‐8RB, and one for the chemoattractant C5a (C5aR) have been cloned. These membrane proteins are members of the rhodopsin superfamily of G‐protein coupled seven‐transmembrane segment receptors. New monoclonal antibodies (mAb) directed against the deduced N‐terminal sequences of the IL‐8RA (mAb SE2) and IL‐8RB (mAb HC2) were generated to determine the IL‐8R expression on human blood leukocytes and two human myeloid cell lines. The C5aR expression was detected using the mAb W17/1. Approximately 107000 C5aR, 55000 IL‐8RA, and 25000 IL‐8RB molecules per cell could be detected on human granulocytes by flow cytometric analysis. On peripheral blood monocytes, 42000 C5aR molecules/cell and 3000 IL‐8RB molecules/cell were expressed. However, we were unable to quantitate IL‐8RA expression, which was detectable but below 2500 molecules per cell and thus outside the standard range for the quantitation of receptor molecules by flow cytometry. On AML‐193 cells, only the IL‐8RB was constitutively expressed, whereas on HL‐60 cells, we could not detect expression of any of the three receptors. Vitamin D3 (250 ng/ml, 7 days), which has been shown to induce differentiation of AML‐193 and HL‐60 cells into the monocytic phenotype, led to an up‐regulation of IL‐8RB and C5aR in both cell lines in the absence of any expression of IL‐8RA. In contrast, all‐trans retinoic acid (0.1 μM, 7 days), which induces differentiation into the granulocytic phenotype, led to an up‐regulation of IL‐8RB in AML‐193 cells and to an expression of IL‐8RB and C5aR in HL‐60 cells. Again, neither cell line expressed IL‐8RA. These findings suggest that regulation of IL‐8RA expression differs from that of its IL‐8RB homolog and may be a late event in leukocyte maturation.
Epitope cleavage byLeishmaniaendopeptidase(s) limits the efficiency of the exogenous pathway of major histocompatibility complex class I‐associated antigen presentation
Authors: Garcia Maria R. , Graham Shek , Harris Robert A. , Beverley Stephen M. , Kaye Paul M.
Published: March 2007

The activation of CD8+T cell responses is commonplace during infection with a number of nonviral pathogens. Consequently, there has been much interest in the pathways of presentation of such exogenous antigens for major histocompatibility complex class I‐restricted recognition. We had previously shown thatLeishmaniapromastigotes transfected with the ovalbumin (OVA) gene could efficiently target OVA to the parasitophorous vacuole (PV), with subsequent recognition by class II‐restricted T cells. We now report the results of studies aimed at evaluating the PV as a route of entry into the exogenous class I pathway. Bone marrow‐derived macrophages can present soluble OVA (albeit at high concentrations) to the OVA257–264‐specific T cell hybridoma 13.13. In contrast, infection with OVA‐transfectedLeishmaniapromastigotes failed to result in the stimulation of this hybridoma. This appeared unrelated to variables such as antigen concentration, parasite survival, and macrophage activation status. These results prompted an analysis of the effects of promastigotes on class I peptide binding using RMA‐S cells and OVA257–264. Our data indicate that the major surface protease ofLeishmania, gp63, inhibits this interaction by virtue of its endopeptidase activity against the OVA257–264peptide. The data suggest that this activity, if maintained within the PV, would result in loss of the OVA257–264epitope. Although we can therefore draw no conclusions from these studies regarding the efficiency of the PV as a site of entry of antigen into the exogenous class I pathway, we have identified a further means by which parasites may manipulate the immune repertoire of their host.
Activation and activation‐induced death of human tonsillar B cells and Burkitt lymphoma cells: lack of CD95 (Fas/APO‐1) ligand expression and function
Authors: Daniel Peter T. , Oettinger Ulrich , Mapara Markus Y. , Bommert Kurt , Bargou Ralf , Dörken Bernd
Published: March 2007

Activation of T cells was shown to up‐regulate the Fas ligand (FasL) which binds to the CD95 (APO‐1/Fas) antigen and mediates activation‐induced cell death (AICD) of activated T cells and T lymphoma cells. A recent report showed that mouse B cells express the FasL upon activation with lipopolysaccharide (LPS). We therefore asked whether activation of human B cells induces expression of FasL and whether AICD is mediated, as in T cells, through autocrine production of the FasL. We used human tonsillar B cells and Burkitt lymphoma cell lines which were activated by CD40 ligand, surface (s)IgM cross‐linking, or LPS. Northern and Western blot analysis failed to detect FasL during B cell activation or AICD of both normal and malignant B cells. Low‐level expression of FasL was detected by reverse transcriptase‐polymerase chain reaction. Functional experiments, however, showed that FasL is not functionally expressed upon activation. IgM‐mediated AICD in the tonsillar or Burkitt lymphoma B cells could not be inhibited by FasL blocking. Thus, our data show that, in contrast to T cells, activation of normal or malignant human B cells does not lead to functional FasL expression.
CD43 (leukosialin, sialophorin) expression is differentially regulated by retinoic acids
Authors: Babina Magda , Weber Sylvia , Henz Beate M.
Published: March 2007

CD43 (leukosialin, sialophorin), a cell‐surface associated mucin that is constitutively expressed at high levels on most leukocytes, is thought to be involved in cell activation and adhesion. We here provide evidence that the vitamin A metabolites all‐trans and 13‐cis retinoic acid up‐regulate CD43 on human leukemic (HMC‐1) mast cells, as determined by flow cytometry, Western blot analysis, and by semiquantitative reverse transcriptase‐polymerase chain reaction. Enhanced CD43 expression was accompanied by a strong increase in anti‐CD43‐mediated, LFA‐1‐dependent homotypic aggregation of HMC‐1 cells, demonstrating that newly synthesized CD43 is functionally active in transmitting signals across the plasma membrane which result in enhanced cellular adhesion. CD43 expression was also enhanced in response to retinoic acids on isolated human skin mast cells and human monocytes, but not on cells of the basophilic cell line KU‐812 and promyelocytic HL‐60 cells, indicating that these agents might act in a cell‐type specific manner. These combined result point to a novel aspect in the regulation of CD43. Possibly, vitamin A metabolites act directly on the CD43 gene, since putative retinoic acid response elements have been detected within its regulatory regions.
Resistance of cultured peripheral T cells towards activation‐induced cell death involves a lack of recruitment of FLICE (MACH/caspase 8) to the CD95 death‐inducing signaling complex
Authors: Peter Marcus E. , Kischkel Frank C. , Scheuerpflug Christian G. , Medema Jan Paul , Debatin Klaus‐Michael , Krammer Peter H.
Published: March 2007

Phytohemagglutinin‐activated peripheral CD95+ T cells (day 1 T cells) are resistant to CD95‐mediated apoptosis. After prolonged interleukin‐2 treatment, these T cells become CD95‐mediated apoptosis‐sensitive (day 6T cells). To elucidate the molecular mechanism of apoptosis resistance, day 1 and day 6 T cells were tested for formation of the CD95 death‐inducing signaling complex (DISC). DISC‐associated active Fas‐associated DD protein (FADD)‐like interleukin‐1β‐converting enzyme‐like protease (FLICE) also referred to as MACH/caspase 8 was only found in apoptosis‐sensitive day 6 T cells. Further analysis of mRNA and protein expression levels of apoptosis‐signaling molecules FADD, receptor interacting protein, hematopoietic cell protein tyrosine phosphatase, Fas‐associated phosphatase‐1, FLICE, bcl‐2, bcl‐xL, and, bax‐α showed that only the expression level of bcl‐xL correlated with T cell resistance to CD95‐mediated apoptosis (day 1 T cells: bcl‐xhiL; day 6 T cells: bcl‐xloL). In T cells activated in vitro, up‐regulation of bcl‐xL has previously been correlated with general apoptosis resistance. However, the experiments presented suggest that resistance to CD95‐mediated apoptosis in T cells can also be regulated at the level of recruitment of FLICE to the DISC.
Effect of interleukin‐10 on dendritic cell maturation and function
Authors: de Smedt Thibaut , van Mechelen Marcelle , De Becker Geneviève , Urbain Jacques , Leo Oberdan , Moser Muriel
Published: March 2007

The main function of dendritic cells (DC) is to induce the differentiation of naive T lymphocytes into helper cells producing a large array of lymphokines, including interleukin (IL)‐2; interferon‐γ (IFN‐γ), IL‐4, IL‐5 and IL‐10. The potent immunostimulatory properties of DC develop during a process of maturation that occurs spontaneously in vitro. Since IL‐10 has been shown to inhibit Th1 responses, we determined its effect on DC maturation and accessory function. Our data show that DC that have undergone maturation in vitro in the presence of IL‐10, have an impaired capacity to induce a Th1‐type response in vivo, leading to the development of Th2 lymphocytes. Their inability to promote the synthesis of IFN‐γ seems to correlate with a decreased production of IL‐12, an heterodimeric cytokine necessary for optimal generation of Th1‐type cells. These results suggest that IL‐10 skews the Th1/Th2 balance to Th2 in vivo by selectively blocking IL‐12 synthesis by the antigen‐presenting cells that play a role of adjuvant of the primary immune response. The cytokines present in the environment at the presentation step may, therefore, determine the class of the immune response induced by DC in vivo, i.e. Th0 Th1 and/or Th2.
Immunoglobulin lambda light chain orphons on human chromosome 8q11.2
Authors: Frippiat Jean‐Pol , Dard Patricia , Marsh Sharon , Winter Greg , Lefranc Marie‐Paule
Published: March 2007

We have identified two Vλ genes outside the major lambda locus on chromosome 22q11.2, and shown that they reside on chromosome 8q11.2. One gene (Orphéel), hybridizing strongly to the Vλ probes, was sequenced and found to belong to the Vλ8 family; the other gene (Orphée2) only hybridized weakly. Orphée1 was present in all individuals tested (140) from three different populations, and was also found in gorillas. We envisage that these genes were generated by duplication and translocation of the Vλ8a gene (and a Vλ pseudogene) from the major locus, and that this event occurred before the evolutionary divergence of humans and gorillas. As there is no other evidence for Vλ genes outside the major locus, it appears that the human lambda locus has undergone considerably less evolutionary shuffling than either the human light chain kappa locus or the heavy chain locus.
Cleavage of caspase family members by granzyme B: a comparative study in vitro
Authors: Van De Craen Marc , Van Den Brande Ilse , Declercq Wim , Irmler Martin , Beyaert Rudi , Tschopp Jürg , Fiers Walter , Vandenabeele Peter
Published: March 2007

The aspartase granzyme B is one of the major components of the granules involved in cell killing by cytotoxic T lymphocytes. Granzyme B has been shown to activate the apoptotic death pathway in the target cell, and this involves activation of members of the caspase (CASP) protein family. Therefore, activational cleavage of mouse (m) CASP proforms by granzyme B was examined in vitro. CASP can be subdivided in the CASP‐1 (interleukin‐1β‐converting enzyme; ICE) subfamily, the CASP‐2 (Ich1) subfamily, and the CASP‐3 (CPP32) subfamily. Our results reveal that the proforms of the CASP‐3 subfamily members mCASP‐3 and mCASP‐7 are hydrolyzed by granzyme B, while proforms of CASP‐2 and CASP‐1 subfamily members are not directly cleaved. Only one CASP‐3 subfamily member, pro‐mCASP‐6, was not proteolytically cleaved by granzyme B. These results indicate that two members of the CASP‐3 subfamily, but no others, become activated by granzyme B.
A retro‐inverso analog mimicks the cognate peptide epitope of a CD4+ T cell clone
Authors: Bartnes Kristian , Hannestad Kristian , Guichard Gilles , Briand Jean Paul
Published: March 2007

Synthetic analogs of peptide epitopes may activate specific T helper cells, antagonize their antigen receptors, or block recognition by competing for major histocompatibility complex (MHC) class II binding sites. Rationally designed peptides may therefore prove useful as vaccines and for treatment of autoimmune diseases and allergies mediated by CD4+ T cells. However, their susceptibility to proteolytic degradation limits the applicability of conventional peptides in vivo. By contrast, retro‐inverso analogs, in which a native sequence is substituted with D‐amino acids linked with a reversed backbone, resist proteolysis and still maintain the side chain topology of the corresponding natural peptide. We report here that an end group‐modified retro‐inverso analog of the IgG2ab heavy chain allopeptide determinant γ2ab 435–447 was recognized by an I‐Ad‐restricted, γ2ab 435–447‐reactive T cell clone. The pseudopeptide elicited near‐maximal interleukin‐2 responses, although 300‐fold higher concentrations were needed than the native determinant. The weaker antigenicity of the retro‐inverso analog could be fully accounted for by an impaired I‐Ad binding capacity, which might reflect reduced ability of the distorted main chain to form hydrogen bonds with I‐Ad. Glycine substitution at the residue corresponding to the first primary anchor (P1) of the native peptide abrogated I‐Ad binding and antigenicity of the retro‐inverso analog. Thus, the pseudopeptide resembled the native determinant with respect to orientation in the class II binding site, configuration of the epitopic side chains, and the constraints that governed the interactions between a major anchoring side chain and I‐Ad. In conclusion, proteolytically resistant compounds with predefined capacity to interact with MHC class II allelic products and T cell antigen receptors may be designed by retro‐inverso modification of native determinants.
Activation of protein kinase C attenuates early signals in Fas‐mediated apoptosis
Authors: Ruiz‐Ruiz María Del Carmen , Izquierdo Manuel , de Murcia Gilbert , López‐Rivas Abelardo
Published: March 2007

Activation of protein kinase C (PKC) has been reported to inhibit Fas (APO‐1, CD95)‐mediated apoptosis in different cellular systems. Human Jurkat leukemic T cells express the Fas antigen in the cell membrane and undergo apoptosis upon cross‐linking by anti‐Fas monoclonal antibodies (mAb). Cleavage of the apoptosis‐associated protease CPP32 and its substrate poly(ADP‐ribose)polymerase are observed after the engagement of Fas antigen with mAb. In this report, we show that all these effects are substantially inhibited by the activation of PKC with a phorbol ester. Bisindolylmaleimide, an inhibitor of PKC, prevents phorbol ester‐induced down‐regulation of Fas signaling. Inhibition of Fas‐mediated cell death by phorbol ester is also observed in other human leukemic T cell lines. Cross‐linking of Fas antigen by mAb results in the rapid increase in tyrosine phosphorylation of several protein substrates which is further elevated in the presence of the protein tyrosine phosphatase inhibitor, orthovanadate. Furthermore, orthovanadate markedly enhances the cell death response to Fas mAb in different human leukemic T cell lines and human T cell blasts. These effects of orthovanadate on early tyrosine phosphorylation and cell death are clearly diminished by PKC activation. These results strongly suggest that tyrosine phosphorylation is involved in Fas signaling in apoptosis and that PKC plays a negative role in Fas‐mediated apoptosis by counteracting at a very early stage the signals generated following cross‐linking of this receptor.
Macrophages sense pathogens via DNA motifs: induction of tumor necrosis factor‐α‐mediated shock
Authors: Sparwasser Tim , Miethke Thomas , Lipford Grayson , Erdmann Andreas , Häcker Hans , Heeg Klaus , Wagner Hermann
Published: March 2007

Cell surface components of pathogens, such as lipopolysaccharide (LPS), are an important signal for receptor‐mediated activation of immune cells. Here we demonstrate that DNA of gram‐positive and gram‐negative bacteria or certain synthetic oligonucleotides displaying unmethylated CpG‐motifs can trigger macrophages in vitro to induce nuclear translocation of nuclear factor‐xB, accumulate tumor necrosis factor (TNF)‐α mRNA and release large amounts of TNF‐α. In vivo these events culminate in acute cytokine‐release syndrome which includes systemic but transient accumulation of TNF‐α. D‐Galactosamine (D‐GalN)‐sensitized mice succumb to lethal toxic shock due to macrophage‐derived TNF‐α resulting in fulminant apoptosis of liver cells. LPS and a specific oligonucleotide synergized in vivo as measured by TNF‐α‐release, suggesting that macrophages integrate the respective signals. The ability of macrophages to discriminate and to respond to bacterial DNA with acute release of pro‐inflammatory cytokines may point out an important and as yet unappreciated sensing mechanism for foreign DNA.
Dominance of the BV17 element in nickel‐specific human T cell receptors relates to severity of contact sensitivity
Authors: Vollmer Jörg , Fritz Michaela , Dormoy Anne , Weltzien Hans Ulrich , Moulon Corinne
Published: March 2007

Hypersensitivity to nickel (Ni) represents the most common manifestation of contact allergy in humans. The role of metal‐specific T cells in this disease is well established, but the molecular interactions involved in their activation are poorly understood. We examined the T cell receptor (TCR) repertoire in T cells activated with either NiSO4 or NiSO4‐treated human serum albumin from six allergic patients. For the three most hyperreactive donors, we found a strong over‐represention of the TCR BV17 element. TCR sequencing for one of these donors revealed an additional skewing for AV1 as well as a selection for an N region encoded argine at position 95 of the BV17 complementarity determining region (CDR)3. Since Arg is not known to participate in Ni complexing, we suppose that this selection is driven by contacts with peptide rather than nickel. However, the CDR1 of BV17 contains a unique combination of amino acids (HDA) that bears similarities to known motifs in Ni‐binding proteins or peptides. We therefore propose that the severe hypersensitivity reactions found in BV17 over‐expressors may be the result of Ni2+ ions bridging the germ‐line‐encoded BV17 CDR1 loop to corresponding sites in the major histocompatibility complex/peptide complex and thereby creating a superantigen‐like enhancement of weak TCR‐peptide contacts.
Cytotoxic T cell reactivity and HLA‐B35 binding of the variant Plasmodium falciparum circumsporozoite protein CD8+ CTL epitope in naturally exposed Kenyan adults
Authors: Udhayakumar Venkatachalam , Ongecha John M. , Shi Ya‐Ping , Aidoo Michael , Orago A. S. S. , Oloo Aggrey J. , Hawley William A. , Nahlen Bernard L. , Hoffman Stephen L. , Weiss Walter R. , Lal Altaf A.
Published: March 2007

In this study, we have investigated the extent of natural polymorphism in the CD8+ cytotoxic T lymphocyte (CTL) determinant (amino acids 368–390) of circumsporozoite (CS) protein of Plasmodium falciparum field isolates from a holoendemic region of Kenya, and determined how this variation affects the CTL reactivities in clinically immune adults and binding specificities to human histocompatibility leukocyte antigen (HLA)‐B35. Among the eight variant sequences that were found in this region, four were new and not seen in parasites from other geographical regions. When synthetic peptides corresponding to the eight variants were used to test the presence of CTL response in different donors, a different spectrum of CTL reactivity to these variants was noticed. While CTL from some donors recognized the P1 sequence (the most prevalent type of sequence) but not P8 (another major variant), other donors showed a reverse pattern of reactivity. Although none of the donors was able to recognize all the variants, CTL responses to all the eight variant sequences were found in this population. An octamer peptide with P1 sequence KPKDELDY in this polymorphic determinant was known to bind HLA‐B35. When we tested the effect of natural variation in this octamer sequence on HLA‐B35 binding, it became evident that SP13 with D → N substitution retained its binding specificity to HLA‐B35. On the other hand, the SP12 octamer sequence which had two substitutions did not bind HLA‐B35. The most interesting finding was the observation that a D → G substitution at position 374 rescued the binding ability of SP14, which otherwise could not bind to this HLA molecule due to E → Q amino acid substitution at position 372. To our knowledge, this is the first demonstration showing that a natural polymorphism can rescue the binding specificity to an HLA‐class I molecule that was lost due to another natural amino acid substitution. Altogether, these results demonstrate that natural polymorphism in the CS protein affects both the CTL reactivity and the ability to bind to HLA‐B35.
The X‐linked immunodeficiency defect in the mouse is corrected by expression of human Bruton's tyrosine kinase from a yeast artificial chromosome transgene
Authors: Maas Alex , Dingjan Gemma M. , Savelkoul Huub F. J. , Kinnon Christine , Grosveld Frank , Hendriks Rudolf W.
Published: March 2007

Mutations in the gene for Bruton's tyrosine kinase result in the B cell differentiation defects X‐linked agammaglobulinemia in man and X‐linked immunodeficiency in mice. Here we describ the generation of two yeast artificial chromosome (YAC)‐transgenic mouse strains in which high‐level expression of human Btk is provided by endogenous regulatory cis‐acting elements that are present on a 340‐kb transgene, Yc340‐hBtk. The expression pattern of the transgenic human Btk was found to parallel that of the endogenous murine gene. When the Yc340‐hBtk‐transgenic mice were mated onto a Btk‐deficient background, the xid B cell defects were fully corrected: conventional and CD5+ B‐1 B cells were present in normal numbers, serum IgM and IgG3 levels as well as responses to T cell‐independent type II antigens were in the normal ranges. In vivo competition experiments in Btk+/− female mice demonstrated that in the conventional B cell population the Yc340‐hBtk transgene could fully compensate the absence of expression of endogenous murine Btk. We conclude that in the YAC‐transgenic mice Btk is appropriately expressed in the context of native regulatory sequences.
Microsites for immunoglobulin switch recombination breakpoints from Xenopus to mammals
Authors: Mußmann Rainer , Courtet Michèle , Schwager Joseph , Du Pasquier Louis
Published: March 2007

Immunoglobulin (Ig) heavy chain class switch recombination has been studied at the DNA level in a non‐mammalian vertebrate, the amphibian Xenopus. A switch (S) region of about 5 kb has been identified in the JH‐Cμ intron of the Ig heavy chain locus in Xenopus. Sμ contains 23 repeats approximately 150 bp long. Each repeat consists of internal shorter repeats and palindromic sequences, such as AGCT, which they share with mammalian switch regions. A deletion of the μ gene and the joining of the S regions of μ and χ occurs in B cells expressing IgX, one of the two non‐μ isotypes in Xenopus. Sx shows no sequence homology with Sμ and is characterized by 16 and 121 bp repeats and a high frequency of CATG, AGCA and TGCA palindromes. Both IgM and IgX S regions are AT rich and not GC rich like mammalian S regions. Recombination occurs, most of the time, at positions (microsites) where a single‐stranded DNA folding program predicts the transition from a stem to a loop structure. This feature is conserved in most mammalian switch junctions which points to the general existence and involvement of microsites at one step of the determination of the recombination breakpoint. The recombinogenic nature of the switch regions is therefore linked to its structure rather than to its base composition, the repetitive occurrence of palindromes being essential at creating many microsites.
Requirement of a second signal via protein kinase C or protein kinase A for maximal expression of CD40 ligand. Involvement of transcriptional and posttranscriptional mechanisms
Authors: Suárez Ana , Mozo Lourdes , Gayo Abel , Zamorano José , Gutierrez Carmen
Published: March 2007

High levels of CD40 ligand (CD40L) protein expression are induced on native T cells by increasing the intracellular Ca2+ concentration. In the present study we have shown that ionomycin induces CD40L gene transcription leading to mRNA accumulation which translates to high levels of protein expression. Conversely, agents which increase the intracellular levels of cyclic AMP (cAMP), such as prostaglandin E2 (PGE2) or dibutyryl cyclic AMP (dbcAMP), were unable to induce CD40L expression on T lymphocytes. Cell activation by phorbol 12‐myristate 13‐acetate (PMA) treatment had a slight effect on increasing CD40L mRNA and protein levels. However, PMA and dbcAMP synergized with ionomycin to significantly increase and to prolong the CD40L expression. Nuclear run‐on assays revealed that PMA, but not dbcAMP, increased threefold the CD40L gene transcription rate induced by ionomycin. This effect was independent of de novo protein synthesis. In addition, at a posttranscriptional level, both reagents synergized with the Ca2+ ionophore to prolong the CD40L mRNA half‐life by a mechanism which was also independent of de novo protein synthesis. Moreover, when transcription was blocked with actinomycin D, an increment of the CD40L transcript levels induced by PMA or dbcAMP on ionomycin‐treated cells was observed in the presence of cycloheximide. This probably means that newly synthesized protein may contribute to the CD40L mRNA destabilization. In summary, these data show that PMA and dbcAMP synergized with ionomycin to increase the CD40L mRNA and protein levels. The up‐regulatory effect of PMA was accomplished at a transcriptional and posttranscriptional level, whereas dbcAMP exerted its synergistic effect exclusively at a posttranscriptional level.