bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and regulation of monocyte chemoattractant protein‐1 by human eosinophils
Authors: Izumi Shinyu , Hirai Koichi , Miyamasu Misato , Takahashi Yuichi , Misaki Yoshikata , Takaishi Toshiaki , Morita Yutaka , Matsushima Kouji , Ida Nobuo , Nakamura Haruji , Kasahara Tadashi , Ito Koji
Published: March 2007

Several recent studies have identified eosinophils as a cellular source of various cytokines, indicating that eosinophils play not only an effector role, but also a regulatory role within the allergic inflammatory cell network. In this study, we demonstrate that eosinophils can generate and secrete monocyte chemoattractant protein‐1 (MCP‐1), a prototype of C‐C chemokines. Eosinophils generated immunoreactive MCP‐1 in response to such diverse stimuli as C5a, formylmethionyl‐leucyl‐phenylalanine (FMLP) and ionomycin, but MCP‐1 production was not induced by interleukin (IL)‐1 or tumor necrosis factor‐α. C5a‐ and FMLP‐induced eosinophil MCP‐1 production was absolutely dependent on pretreatment with cytochalasin B. Eosinophils elaborated significantly more MCP‐1 than neutrophils. Immunoreactive MCP‐1 was detected at 6 h of incubation with C5a or FMLP. Expression of MCP‐1 mRNA reached a maximum within the first 3 h after stimulation and then declined rapidly to a very low and stable level by 18 h. Pretreatment with IL‐5 markedly amplified C5a‐induced MCP‐1 production, and the enhancement occurred at the pretranslational level. Eosinophilactive chemokines such as eotaxin failed to induce MCP‐1 generation, even when eosinophils were primed by IL‐5. Since MCP‐1 exerts a potent histamine‐releasing effect on human basophils, our results indicate that eosinophils may regulate basophil mediator release with possible consequent contribution to the pathogenesis of allergic inflammation via a paracrine mechanism.
A DR17‐restricted T cell epitope from a secreted Mycobacterium tuberculosis antigen only binds to DR17 molecules at neutral pH
Authors: Geluk Annemieke , Van Meijgaarden Krista E. , De Vries Rene R. P. , Sette Alessandro , Ottenhoff Tom H. M.
Published: March 2007

The assembly of peptide‐major histocompatability class II complexes in vitro is accelerated at low pH, comparable to that found in the intracellular compartments of metabolically active antigen‐presenting cells (APC). Mycobacteria such as Mycobacterium tuberculosis reside in phagosomes with only mildly acidic pH. Therefore, we investigated the pH dependency of peptide‐HLA‐DR binding for several T cell epitopes of mycobacterial proteins, focussing particularly on well‐defined, immunodominant HLA‐DR17(3)‐restricted T cell epitopes: peptide (p) 3–13 from the cytoplasmic 65‐kDa heat shock protein of M. tuberculosis/M. leprae, and peptide 56–65 from the secreted 30/31‐kDa protein from M. tuberculosis/M. leprae. p3–13 bound to purified, cell‐free DR17 under both acidic and neutral conditions. Four other, unrelated DR17‐binding peptides showed the same pH‐dependent binding characteristics as p3–13. p56–65, however, only bound to purified DR17 at pH 7 but not at all at pH 4.5. These DR17 peptide binding data were confirmed in cell‐bound DR17, in T cell stimulation assays in which fixed APC were peptide‐pulsed at acidic or neutral pH before addition of peptide‐specific DR17‐restricted T cells. As far as we are aware, p56–65 is the only human T cell epitope binding to HLA exclusively at neutral pH. The binding characteristics of p56–65 may reflect dominant processing in alternative, less acidic vacuolar compartments specifically related to the generation of epitopes from (secreted) mycobacterial proteins. The observation that p56–65 is an immunodominant epitope for anti‐mycobacterial T cells suggests the relevance of such novel processing compartments in T cell‐mediated immunity.
Interleukin‐12 promotes a chronic intestinal nematode infection
Authors: Bancroft Allison J. , Else Kathryn J. , Sypek Joseph P. , Grencis Richard K.
Published: March 2007

Resistance and susceptibility to the intestinal parasite Trichuris muris has been shown to be due to a dominant T helper 2 (Th2) and a dominant Th1 response, respectively. The factors determining the initial polarization of the immune response remain largely unresolved, although the cytokine environment at the time of antigen presentation clearly plays an essential role. Interleukin (IL)‐12, a cytokine produced mainly by macrophages, dendritic cells, and other monocytes has been shown to be important in driving a strong Th1 response by stimulating the production of interferon (IFN)‐γ from natural killer and Th0 cells and therefore forms a link between the innate and adaptive immune system. IL‐12 has been shown to play an important role in resistance to a number of intracellular pathogens, including Listeria and Leishmania. It has also been proposed as an anti‐tumor agent and for use in the treatment of HIV. Conversely, IL‐12 has been shown to prolong the survival of Nippostrongylus brasiliensis and to accelerate autoimmunity. Our studies demonstrate that by driving a strong Th1 response, IL‐12 promotes chronic T. muris infection when given to normally resistant BALB/K mice. Parasite‐specific IgG2a, a Th1 parameter of infection, was greatly up‐regulated, whereas some Th2 parameters of infection were down‐regulated. IL‐12 treatment could be delayed until 1 week after infection had started and still promote a strong Th1 response. The actions of IL‐12 in promoting a chronic infection were IFN‐γ dependent as an anti‐IFN‐γ mAb abrogated the effects of IL‐12.
The Oka blood group antigen is a marker for the M6 leukocyte activation antigen, the human homolog of OX‐47 antigen, basigin and neurothelin, an immunoglobulin superfamily molecule that is widely expressed in human cells and tissues
Authors: Spring Frances A. , Holmes Christopher H. , Simpson Karen L. , Mawby William J. , Jules Mattes M. , Okubo Yasuto , Parsons Stephen F.
Published: March 2007

The high‐frequency blood group antigen Oka is carried on a red cell membrane glycoprotein (gp) of 35–69 kDa that is widely distributed on malignant cells of different origins. Immunostaining of hemopoietic cells and a range of normal human tissues demonstrated a wide distribution of the Oka gp that appears to be nonlineage‐restricted, although certain tissues show differentiation‐related expression. Oka gp was purified from red cell membranes by immunoaffinity chromatography using mAb A103 and amino acid sequence analysis was performed. The N‐terminal 30 amino acids are identical to the predicted sequence of M6 leukocyte activation antigen (M6), a member of the Ig superfamily (IgSF) with two IgSF domains. There are homologs in rat (MRC OX‐47 or CE9), in mouse (basigin or gp42), and in chicken (HT7 or neurothelin). The molecular basis of the Oka mutation was established by sequencing M6 cDNA derived from normal and Ok(a‐) EBV‐transformed B cell lines. A point mutation in the translated portion of M6 cDNA, G331 AG → AAG gives rise to a predicted E92 → K amino acid change in the first Ig‐like domain of the Ok(a‐) form of the protein. Transfection of mouse NS‐0 cells with normal or Ok(a‐) cDNA confirmed the identity of the protein and only the Ok(a‐) transfectants failed to react with monoclonal anti‐Oka Ab.
Comparative analysis of αβ and γδ T cell activation by Mycobacterium tuberculosis and isopentenyl pyrophosphate
Authors: Wesch Daniela , Marx Sibylle , Kabelitz Dieter
Published: March 2007

Phosphorylated nonpeptide compounds have recently been identified as potent mycobacteria‐derived ligands for human Vγ9/Vδ2‐expressing γδ T cells. Crude mycobacterial extracts also contain protein antigens which stimulate CD4 αβ T cells to produce growth factors that are used by γδ T cells for clonal expansion. We have investigated the dynamics in vitro of expansion of CD4 T cells and Vγ9 cells in cultures of peripheral blood mononuclear cells stimulated with synthetic isopentenyl pyrophosphate (IPP) in the absence or presence of additional stimuli. The results indicated that following stimulation with IPP, γδ T cells express CD25 and CD69 antigens, but fail to proliferate unless growth factors are provided exogenously or endogenously through activation of CD4 T cells by additional stimuli such as tetanus toxoid, alloantigen, or superantigens. Furthermore, the presence of antigen presenting cells are required for expansion of γδ T cells. In response to IPP stimulation, purified CD4 T cells neither express CD25 or CD69, nor do they proliferate even in the presence of exogenous IL‐2. Apart from IL‐2, IL‐15 and, less efficiently, IL‐4, IL‐7, and IL‐12 can contribute to cellular expansion of IPP‐reactive Vγ9 cells. Together, the results demonstrate that peripheral blood γδ T cells proliferate in response to IPP only if CD4 T cells are simultaneously activated by an additional stimulus. This mechanism provides a tight control of the reactivity of γδ T cells towards phosphorylated nonpeptide antigens.
Role of ligands in the activation of LFA‐1
Authors: Buckley Christopher D. , Ferguson Elaine D. , Littler Amanda J. , Bossy David , Simmons David L.
Published: March 2007

Lymphocyte function‐associated‐antigen‐1 (LFA‐1) is able to bind selectively to its ligands intercellular adhesion molecules 1 and 3 (ICAM‐1 and ICAM‐3), suggesting that LFA‐1 can exist in distinct ligand‐specific binding states. In the case of ICAM‐1, apart from ligand itself and the recently cloned molecule cytohesin‐1, the natural physiological regulators of LFA‐1‐mediated binding to ICAM‐1 are unknown. We have investigated the role of ligands (ICAM‐1 and ICAM‐3) in LFA‐1 activation by using ICAM‐blocking monoclonal antibodies and a fixation protocol for “freezing” LFA‐1 on the surface of cells after prior exposure to ICAM‐1 and ICAM‐3. These studies not only confirm that LFA‐1 exists in distinct ICAM‐specific activation states, but also demonstrate that ICAM‐1 plays a role in the activation of LFA‐1 binding to ICAM‐3.
Differential generation of class I H‐2D‐ versus H‐2K‐restricted cytotoxicity against a demyelinating virus following central nervous system infection
Authors: Lin Xiaoqi , Pease Larry R. , Rodriguez Moses
Published: March 2007

Despite the fact that both H‐2K and D molecules are up‐regulated in the central nervous system (CNS) following Theiler's murine encephalomyelitis virus (TMEV) infection, resistance in this virus model of multiple sclerosis maps exclusively to D. To address this paradox, we examined the ability of the K and D molecules to present viral antigens to cytotoxic T lymphocytes (CTL). Whereas no virus‐specific CTL were detected in the CNS of susceptible B10.Q and B10.S mice 7 days post‐infection, D‐restricted CTL were identified readily in the CNS of resistant B10 animals. There was no evidence of K‐restricted CTL in the CNS of B10 mice at day 7 post‐infection. The presence of both K‐ and D‐restricted virus‐specific CTL in the spleen of immunized B10 mice demonstrates that the exclusive use of D molecules by CTL in the CNS of mice 7 days post‐infection is not due to the inability of the K molecules to present viral peptides to lymphocytes. We conclude that the prominent role of the D locus in determining resistance or susceptibility to TMEV‐induced demyelination is determined by factors governing the regulation of the immune response, and not by the presence or absence of CTL precursors capable of recognizing viral peptides presented by the K and D antigen‐presenting molecules, or by differences in the ability of the K and D molecules to present viral peptides.
Engagement of the B lymphocyte antigen receptor induces presentation of intrinsic immunoglobulin peptides on major histocompatibility complex class II molecules
Authors: Bartnes Kristian , Hannestad Kristian
Published: March 2007

By means of the clonotypic variable region, the immunoglobulin (Ig) is a tumorspecific antigen on B cell neoplasms. We report that engagement of the B cell antigen receptor (BcR) promotes presentation of peptides derived from the B cell's intrinsic Ig to major histocompatibility complex (MHC) class II‐restricted T cells. Thus, anti‐Ig endowed normal, ex vivo B lymphocytes from H‐2d, Ig constant heavy chain allotype b (IgCHb) mice with the capacity to stimulate an I‐Ad‐restricted T cell clone which recognizes the γ2ab 435–451 allopeptide. The corresponding self γ2aa peptide is cryptic and 6000‐fold less antigenic than the γ2ab allopeptide. Even so, the syngeneic B cell lymphoma A20 which expresses surface (s) IgG2aa, was also recognized by the T cells after BcR ligation. Thus, anti‐Ig triggered the disclosure of a cryptic tumor antigen determinant. We propose that autoantigens, by engaging the BcR of self‐reactive B cells, induce presentation of intrinsic Ig peptides to which the T helper cell (Th) repertoire is not tolerant. In this way, B cells with anti‐self potential may be activated without Th recognition of nominal autoantigen.
Mimotopes of polyreactive anti‐DNA antibodies identified using phage‐display peptide libraries
Authors: Sibille Pierre , Ternynck Thérèse , Nato Faridabano , Buttin Gérard , Strosberg Donny , Avrameas Alexandre
Published: March 2007

Three monoclonal IgG2a anti‐DNA polyreactive autoantibodies, derived from lupus‐prone mice (NZB X NZW)F1, were studied by surface plasmon resonance (BIAcore) analysis using three different synthetic double‐stranded (ds) oligonucleotides of 25, 30, and 25 base pairs (bp). These monoclonal antibodies (mAb) exhibited dissociation rate constants (koff) ranging from 0.0001 (mAb F14.6 and F4.1) to 0.01/s (mAb J20.8) and kon ranging from 2 × 105 to 2 × 106/M/s. The screening of a constrained random peptide library displayed on M13 bacteriophages on these mAb allowed the determination of the specific consensus motifs (mimotopes) for mAb F14.6 and J20.8, but not for mAb F4.1. No cross‐reaction was observed between F14.6‐ and J20.8‐specific peptides (and vice versa). Binding of all phages selected on F14.6 was inhibited with 700 ng/ml soluble DNA. The binding of one group of peptides selected on J20.8 was inhibited by 400 ng/ml soluble DNA, of a second group by 2500 ng/ml, while binding of a third group could not be inhibited. The determined consensus sequences do not match with known sequences. Peptides specific for F14.6 share negative charges and aromatic rings that may mimic a DNA backbone, while peptides selected with J20.8 do not bear any negative charge, implying a different kind of molecular recognition, for example hydrogen or salt bonds. The peptides selected on J20.8 also bind serum antibodies from human patients with systemic lupus erythematosus. In addition, BALB/c mice immunized with some of the selected phages exhibit high serum titers of IgG3 anti‐dsDNA antibodies, further supporting the hypothesis that peptide epitopes may mimic an oligonucleotide structure.
Analysis of the requirement for β2‐microglobulin for expression and formation of human CD1 antigens
Authors: Bauer Anton , Hüttinger Robert , Staffler Günther , Hansmann Cornelia , Schmidt Walter , Majdic Otto , Knapp Walter , Stockinger Hannes
Published: March 2007

Human CD1 form a group of nonpolymorphic leukocyte surface molecules with homology to major histocompatibility complex (MHC) proteins. Recent findings in human and in mouse demonstrate the capacity of CD1 molecules to present nonpeptide components like lipids or lipoglycans as well as peptides. We studied the involvement of β2‐microglobulin (β2m) in expression of the classic human CD1 proteins CD1a, CD1b, and CD1c. The β2m‐deficient human melanoma cell line FO‐1 was transiently transfected with either CD1a, CD1b, or CD1c DNA alone, or in combination with β2m using the adenovirus‐enhanced receptor‐mediated transfer infection system. Only co‐transfection of FO‐1 cells with CD1 + β2m resulted in the detection of CD1 Ag by monoclonal antibodies (mAb). This indicated that CD1 mAb recognized determinants are dependent on β2m and raised the question whether β2m‐free forms of CD1 can be expressed. Therefore, to visualize CD1 molecule expression independently of β2m, we expressed tagged recombinant forms. A full‐length CD1b construct tagged at the very C terminus with a small peptide was transported to the plasma membrane only when β2m was co‐transfected. β2m involvement in the transport of CD1 was confirmed by expression of soluble forms of CD1a, CD1b, and CD1c in three different cell types. Analogous to tagged full‐length CD1b, secretion of the soluble CD1 constructs was strictly dependent on β2m. The soluble CD1 chimeras were secreted as complexes with endogenous β2m. Thus, similar to its role for MHC class I expression, β2m is essential for processing and surface transport of the classic human CD1 molecules CD1a, CD1b, and CD1c.
Interleukin‐15‐induced maturation of human natural killer cells from early thymic precursors: selective expression of CD94/NKG2‐A as the only HLA class I‐specific inhibitory receptor
Authors: Mingari Maria Cristina , Vitale Chiara , Cantoni Claudia , Bellomo Rosa , Ponte Marco , Schiavetti Francesca , Bertone Stefania , Moretta Alessandro , Moretta Lorenzo
Published: March 2007

Immature postnatal thymocytes were shown to contain precursors which, under suitable culture conditions, give rise to phenotypically and functionally mature natural killer (NK) cells. Here, we analyzed the effect of different cytokines for their ability to induce the expression of HLA class I‐specific inhibitory receptor(s) during the process of NK cell development from immature thymocytes. From thymocyte cell suspensions depleted of CD2+, CD3+, CD4+, CD8+, CD56+, and CD16+ cells, we further removed cells expressing HLA class I‐specific inhibitory receptors including CD94/NKG2‐A, p58.1, and p58.2 by immunomagnetic bead separation. The resulting cells did not contain any of the above NK receptors as determined by immunofluorescence and flow cytometric analysis, as well as by reverse transcriptase polymerase chain reaction (RT‐PCR) amplification using appropriate sets of primers. Although different cytokines have been used, including interleukin (IL)‐7, stem cell factor (SCF), IL‐2, and IL‐15, only IL‐2 or IL‐15 induced cell proliferation when used alone. Moreover, maturation towards CD3− CD56+ cells displaying cytolytic activity against the HLA class I− targets K562 or 221 was detectable in cultures containing IL‐15 used alone or in combination with IL‐7 or SCF. On the other hand, these CD3− CD56+ cell populations did not lyse HLA class I+ target cells, including autologous PHA blasts. Analysis of the expression of the various HLA class I‐specific inhibitory NK receptors revealed the presence of high proportions of CD94/NKG2‐A+ cells, while the NK receptors belonging to the Ig superfamily were undetectable both by immunofluorescence and by RT‐PCR analysis. The expression of CD94/NKG2‐A appeared to be responsible for the inability of cells to lyse HLA class I+ target cells. Thus, addition of anti‐CD94 monoclonal antibodies of IgM isotype resulted in lysis of autologous target cells. The use of 221 cells transfected with different HLA class I alleles as target cells confirmed the broad class I specificity of CD94/NKG2‐A receptor. Our experiments indicate that IL‐15 provides an appropriate stimulus to the expression of CD94/NKG2‐A, but not of other class I‐specific NK receptors in the process of maturation of NK cells from thymocyte precursors.
Thymocytes and RelB‐dependent medullary epithelial cells provide growth‐promoting and organization signals, respectively, to thymic medullary stromal cells
Authors: Naspetti Marianne , Aurrand‐Lions Michel , Dekoning Jenefer , Malissen Marie , Galland Franck , Lo David , Naquet Philippe
Published: March 2007

The thymic medulla is composed of distinct epithelial cell subsets, defined in this report by the reactivity of two novel antibodies, 95 and 29, raised against mouse thymic epithelial cell lines. These antibodies were used to probe the development of medulla in wild‐type or mutant thymuses. In CD3σ‐deficient mice where thymocyte maturation is arrested at the CD4− CD8− stage, few scattered 95+ and 29+ epithelial cells are found. When few mature thymocytes develop as in CD3‐ζ/η mice, expansion and organization of 95+ but not 29+ cells, becomes detectable. In RelB‐deficient mice, T cell maturation proceeds normally but negative selection is inefficient due to the lack of thymic medulla and dendritic cells. Strikingly, 29+ epithelial cells are absent and 95+ medullary epithelial cells are scattered throughout the thymus, intermingling with CDR1+ cortical epithelium. In chimeric mice lacking only dendritic cells, the corticomedullary junction persists and both 95+ and 29+ epithelial cells are localized in the medulla. These results suggest that two types of signals are required for development of thymic medulla. A growth signal depends upon the presence of maturing thymocytes, but organization of the thymic medulla requires the presence of activated 29+ medullary epithelial cells.
Lack of directed Vα14‐Jα281 rearrangements in NK1+ T cells
Authors: Shimamura Michio , Ohteki Toshiaki , Beutner Ulrich , Robson MacDonald H.
Published: March 2007

NK1.1+ T cells are an unusual subset of TCRαβ cells distinguished by their highly restricted Vβ repertoire and predominant usage of an invariant Vα14‐Jα281 chain. To assess whether a directed rearrangement mechanism could be responsible for this invariant α chain, we have analyzed Vα14 rearrangements by polymerase chain reaction and Southern blot in a panel of cloned T‐T hybrids derived from thymic NK1.1+ T cells. As expected a high proportion (17/20) of the hybrids had rearranged Vα14 to Jα281. However, Vα14‐Jα281 rearrangements always occurred on only one chromosome and were accompanied by other Vα‐Ja rearrangements (not involving Vα14) on the homologous chromosome. These data argue that rigorous ligand selection rather than directed rearrangement is responsible for the high frequency of Vα14‐Jα281 rearrangements in NK1.1+ T cells.
On the mechanisms by which transforming growth factor‐β2 alters antigen‐presenting abilities of macrophages on T cell activation
Authors: Takeuchi Masaru , Kosiewicz Michele M. , Alard Pascale , Streilein J. Wayne
Published: March 2007

Peritoneal exudate cells (PEC) incubated with antigen in the presence of transforming growth factor‐(TGF)‐β2 selectively suppress delayed hypersensitivity and IgG2a antibody production when injected intravenously into naive syngeneic recipients. In this study, we have examined in vitro the effects of TGF‐β2 on the antigen presenting abilities of PEC to activate DO11.10 T cells that express a transgenic T cell receptor that recognizes ovalbumin peptide fragment 323–339 in the context of I‐Ad. PEC were pretreated overnight with TGF‐β2, washed extensively, then co‐cultured with DO11.10 T cells in the presence of native OVA or P323–339. We found that TGF‐β2‐treated PEC induced the production of the T helper type 2 (Th2) cytokine, interleukin‐4 (IL‐4), but unlike untreated PEC, were unable to stimulate the Th1 cytokines, IL‐2 and interferon‐γ (IFN‐γ). Furthermore, TGF‐β2 was produced in an autocrine fashion by TGF‐β2‐treated PEC and was responsible for this shift to a Th2 response. This conclusion was supported by the following results. First, TGF‐β2‐treated PEC were found to express much more TGF‐β1 and TGF‐β2 mRNA than untreated PEC. Second, TGF‐β2‐treated PEC secreted large amounts of TGF‐β including its mature form. Third, addition of neutralizing anti‐TGF‐β2 antibodies, but not neutralizing anti‐TGF‐β1 antibodies, restored the ability of antigen‐pulsed, TGF‐β2‐pretreated PEC to stimulate DO11.10 T cells to secrete IL‐2 and IFN‐γ. These results indicate that antigen‐presenting cells that encounter antigen in a TGF‐β‐enriched environment (e.g., in the eye) shift responding naive T cells toward Th2 responses by producing TGF‐β during antigen presentation.
Analysis of HLA class Ib gene expression in male gametogenic cells
Authors: Fiszer Dorota , Ulbrecht Matthias , Fernandez Nelson , Johnson Judith P. , Weiss Elisabeth H. , Kurpisz Maciej
Published: March 2007

We have investigated mRNA expression for nonclassical MHC class I genes (HLA‐E,‐F,‐G) in human gametogenic cells. Testicular tissue was treated by collagenase and the resulting cell suspension was further purified by fractionation on Percoll gradients in a two‐step procedure. Three gametogenic cell fractions were analyzed: purified heterogenous suspension of gametogenic cells, fraction of round spermatids and fraction of elongated spermatids. Total RNA isolated from each cell population was subjected to both reverse transcriptase/polymerase chain reaction and Northern blot analysis using oligonucleotides specific for HLA‐E, ‐F and ‐G. Both method gave similar results. We have found a considerable level of HLA‐E mRNA, very low amounts of reamplified cDNA for HLA‐F and both a complete lack of mRNA and reamplified cDNA for the HLA‐G gene in the analyzed gametogenic cell fractions. Additionally, we have localized HLA‐E molecules on the cells of the adluminal compartment within seminiferous tubules using immunostaining with monoclonal antibodies specific for HLA‐E heavy chain followed by confocal microscopy analysis. The unique expression pattern of HLA class I antigens in the male gonad could play an important role in an efficient protection against an autoimmunological attack toward germ cells.
Crucial role of tumor necrosis factor (TNF) receptor 2 and membrane‐bound TNF in experimental cerebral malaria
Authors: Lucas Rudolf , Juillard Pierre , Decoster Els , Redard Mireille , Burger Danielle , Donati Yves , Giroud Christine , Monso‐Hinard Christine , De Kesel Toon , Buurman Wim A. , Moore Mark W. , Dayer Jean‐Michel , Fiers Walter , Bluethmann Horst , Grau Georges E.
Published: March 2007

Tumor necrosis factor (TNF) has been implicated in the pathogenesis of experimental cerebral malaria (CM), but the respective role of its two types of receptors has not been established. A significant increase in the expression of TNF‐receptor 2 (TNFR2, p75), but not of TNFR1 (p55), was found on brain microvessels at the time of CM in susceptible animals. Moreover, mice genetically deficient for TNFR2 (Tnfr2°) were significantly protected from experimental CM, in contrast to TNFR1‐deficient (Tnfr1°) mice, which were as susceptible as wild‐type mice. To identify the factors involved in the protection from CM conferred by the lack of TNFR2, we assessed in both knockout and control mice the serum concentrations of mediators that are critical for the development of CM, as well as the up‐regulation of intercellular adhesion molecule‐1 (ICAM‐1) in the brain microvessels. No significant difference in serum levels of TNF and interferon‐γ was found between infected wild‐type and Tnfr1° or Tnfr2° mice. Interestingly, the pronounced ICAM‐1 up‐regulation and leukocyte sequestration, typically occurring in brain microvessels of CM‐susceptible animals, was detected in infected control and Tnfr1° mice – both of which developed CM – whereas no such ICAM‐1 up‐regulation or leukocyte sequestration was observed in Tnfr2° mice, which were protected from CM. Making use of microvascular endothelium cells (MVEC) isolated from wild‐type, Tnfr1° or Tnfr2° mice, we show that soluble TNF requires the presence of both TNF receptors, whereas membrane‐bound TNF only needs TNFR2 for TNF‐mediated ICAM‐1 up‐regulation in brain MVEC. Thus, only in MVEC lacking TNFR2, neither membrane‐bound nor soluble TNF cause the up‐regulation of ICAM‐1 in vitro. In conclusion, these results indicate that the interaction between membrane TNF and TNFR2 is crucial in the development of this neurological syndrome.
T cell repertoire formation displays characteristics of qualitative models of thymic selection
Authors: Elliott James I.
Published: March 2007

The use of T cell receptor elements varies between mouse strains, reflecting a balance between positive and negative selection. The presence of H‐2E biases Vα and Vβ usage through major histocompatibility class II isotype preferences of V elements, and mammary tumor virus‐dependent, negative selection. Quantitative models of thymic selection predict that negative selection equates to ‘excess’ positive selection, whereas qualitative models suggest that positive and negative selection are opposing forces. This report attempts to distinguish between the models by assessing whether, at the level of the T cell repertoire, positive and negative selection have quantitative or qualitative characteristics. The data show that the effect of bearing Vα and Vβ regions which are both preferentially (or negatively) selected in the presence of H‐2E is additive or synergistic, whilst positive stimuli counteract negative ones. The data thus provide support for qualitative models of thymic selection.
PU.1 negatively regulates the CD11c integrin gene promoter through recognition of the major transcriptional start site
Authors: López‐Rodríguez Cristina , Corbí Angel L.
Published: March 2007

CD11c integrin expression is restricted to myeloid cells and activated B lymphocytes, mainly through the collaborative action of Sp1 and members of the AP‐1 and C/EBP transcription factor families on the proximal region of the CD11c gene promoter. While analyzing the role of an initiator‐like sequence at the major transcriptional start site, an inverted consensus GGAA Ets binding site was identified as a negative regulatory element whose disruption increases the activity of the CD11c promoter. The GGAA element was specifically recognized by PU.1 in THP‐1 monocytic cells and by PU.1 and GABP‐related proteins in U937 promonocytic cells. Mutational analysis indicated that PU.1 recognition depends not only on the GGAA consensus element but also on flanking sequences. The functional relevance of PU.1 binding was assayed in transactivation experiments in HeLa cells, where PU.1 co‐expression led to a significant decrease in the activity of the CD11c promoter, demonstrating that PU.1 inhibits the activity of the CD11c promoter through a PU.1 binding site located at the major transcriptional start site (PU1‐5). The inhibitory action of PU.1 on CD11c is in contrast with its positive regulatory effect on the CD11b and CD18 integrin gene promoters, which might contribute to the differentially regulated expression of CD11b/CD18 and CD11c/CD18 during monocyte extravasation and terminal maturation. In addition, since PU.1 transcriptional activity correlates with macrophage proliferation, PU.1 might modulate CD11c gene transcription according to the proliferative state of the cell.
The leukocyte/neuron cell surface antigen OX2 binds to a ligand on macrophages
Authors: Preston Sandy , Wright Gavin J. , Starr Karen , Barclay A. Neil , Brown Marion H.
Published: March 2007

The OX2 membrane glycoprotein contains two immunoglobulin superfamily (IgSF) domains and seems likely to interact with other cell surface proteins. A soluble chimeric protein with the two IgSF domains of OX2 engineered onto domains 3 + 4 of rat CD4 antigen was expressed. To detect possible weak interactions, the chimeric protein was coupled to fluorescent covaspheresTM to provide a highly avid display of OX2. The OX2 covaspheres bound macrophages but not other cell types. The specificity of the interaction was demonstrated by blocking with Fab fragments of the OX2 monoclonal antibody (mAb). A new mAb, MRC OX88, was raised against macrophages which also blocked the interaction and presumably recognizes the ligand. The epitope for the MRC OX2 mAb and a site for ligand binding were mapped to domain 1 by site‐directed mutagenesis. The OX2 antigen is present on thymocytes, some lymphocytes, neurons and endothelial cells; thus, it has the potential to mediate interactions between these cell types and macrophages.
CD9, but not other tetraspans, associates with the β1 integrin precursor
Authors: Rubinstein Eric , Poindessous‐Jazat Virginie , Le Naour François , Billard Martine , Boucheix Claude
Published: March 2007

The molecules of the tetraspan superfamily have two unequal extracellular domains separated by four transmembrane (TM) domains. These molecules are associated on the cell surface with each other and with other partner molecules, in particular β1 integrins. We now show that CD9 associates with the precursor of the β1 integrin (preβ1). This association is detected as early as 15 min after metabolic labeling, and the use of Brefeldin A demonstrates that it does not require Golgi modifications of either CD9 or integrin β1. The specificity of this interaction is demonstrated by the fact that other tetraspans, CD63, CD81, and CD82, do not associate with preβ1, and CD9 does not associate with immature human histocompatibility leukocyte antigen class I. In order to localize the region of CD9 responsible for the association with the β1 integrin, we have generated two reciprocal chimeric CD9/CD82 molecules with the junction localized just after the third TM region. The large extracellular loop of CD9 or the fourth TM domain, or both, appear to be sufficient to mediate an association with the mature integrin with a high efficiency, compared to CD82. By contrast, association with preβ1 requires at least two regions of the molecule. Mutation of CD9 at the consensus site of the tetraspan superfamily, localized between the second and the third TM domain, did not impair the co‐precipitation of preβ1. Finally, because preβ1 associates with calnexin, we have investigated a possible association of CD9 with this chaperone molecule. CD9 associates with calnexin independently of its association with the β1 integrin, suggesting that calnexin could be involved in the processing of CD9.
Identification of a Stat‐6‐responsive element in the promoter of the human interleukin‐4 gene
Authors: Curiel Rafael E. , Lahesmaa Riitta , Subleski Jeff , Cippitelli Marco , Kirken Robert A. , Young Howard A. , Ghosh Paritosh
Published: March 2007

Interleukin (IL)‐4 is an immunomodulatory cytokine produced by a number of cell types including T cells, basophils, and mast cells. This pleiotropic cytokine has a number of immunoregulatory functions; however, the molecular mechanisms controlling the transcription of this gene are not yet completely understood. Several studies have implicated a possible autoregulatory mechanism for its own expression. Here, we have identified a Stat‐6‐responsive element (Stat‐6RE) in the promoter of the human IL‐4 gene. Utilizing electrophoretic mobility shift analysis, we have demonstrated the presence of two specific IL‐4‐responsive DNA‐protein complexes in nuclear extracts of both human Th1 and Th2 clones. Phytohemagglutinin‐blasted peripheral blood T cells also generated an inducible complex in response to stimulation with IL‐4 and the IL‐4‐like cytokine IL‐13. Transient transfection of the murine pre‐B cell line BA/F3 stably transfected with the full‐length human IL‐4 receptor α chain demonstrated the ability of multicopy Stat‐6RE to initiate transcription from a heterologous promoter upon IL‐4 or IL‐13 stimulation. These results indicate a possible autocrine mechanism for the regulation of IL‐4 gene transcription through the Stat‐6RE as well as a possible mechanism for IL‐13 regulation of the human IL‐4 promoter.
Functional analysis of immunoreceptor tyrosinebased activation motif (ITAM)‐mediated signal transduction: the two YxxL segments within a single CD3ζITAM are functionally distinct
Authors: Sunder‐Plassmann Raute , Lialios Faye , Madsen Mari , Koyasu Shigeo , Reinherz Ellis L.
Published: March 2007

Functional analysis of the immunoreceptor tyrosine‐based activation motif (ITAM) derived from the membrane‐proximal ITAM of CD3ζ demonstrates that mutations at either the tyrosine or leucine residues in the N‐terminal YxxL segment of the ITAM abolish all signal transduction functions of this ITAM. In contrast, mutations at the tyrosine or leucine residues in the C‐terminal YxxL segment abrogate signals for interleukin (IL)‐2 production but do not prevent tyrosine phosphorylation of the N‐terminal tyrosine of the ITAM, lck association with the ITAM, activation of phospholipase C‐γl or calcium mobilization. Cross‐linking of chimeric receptors containing a C‐terminal YxxL leucine mutation induces tyrosine phosphorylation of ZAP70 but without stable binding to the phosphorylated ITAM. These results indicate that the two YxxL segments in an ITAM are functionally distinct and that both are essential for ZAP70 binding and IL‐2 production. Furthermore, tyrosine phosphorylation of ZAP70 per se is not sufficient to trigger the downstream events leading to IL‐2 production. Substitution of an alanine for the bulky side chain at the Y+1 position of the N‐terminal YxxL segment reduces the receptor cross‐linking requirement necessary to achieve cellular activation and the absolute dependence on lck in this process. Our results reveal that both the number of ITAM as well as the specific amino acid residues within a single ITAM determine the extent of chimeric receptor cross‐linking required to trigger tyrosine phosphorylation‐dependent signaling events.
Differentiation of CD4highCD8low coreceptor‐skewed thymocytes into mature CD8 single‐positive cells independent of MHC class I recognition
Authors: Barthlott Thomas , Kohler Hubertus , Pircher Hanspeter , Eichmann Klaus
Published: March 2007

Thymocytes with a CD4hiCD8lo coreceptor‐skewed (CRS) phenotype have been shown to contain precursors for CD8 single‐positive (SP) thymocytes, in addition to precursors for CD4 SP cells. The selection mechanisms that stimulate CD4hiCD8lo cells to revert to the CD8 lineage are not known. Mice transgenic (tg) for the major histocompatibility complex (MHC) class I‐restricted P14 T cell receptor (TCR), on the H‐2bm13 background, generate a large number of CD4hiCD8lo CRS thymocytes. We analyzed the developmental potential and the differentiation requirements of the CD4hiCD8lo population of these mice. Using reaggregate thymic organ cultures (RTOC), we observed that these cells efficiently and almost exclusively differentiate into CD8 SP cells. Differentiation occurred independent of whether or not the MHC haplotype of the thymic stroma corresponds to the MHC restriction of the tg TCR. Loss of CD4 was independent of thymic stroma, up‐regulation of CD8 to full levels was dependent on thymic stroma but independent of MHC haplotype. After trypsin treatment and overnight incubation, these CRS cells re‐expressed CD8 but failed to re‐express CD4, indicating that they are in the process of terminating CD4 synthesis. CD8 SP cells derived from the CRS cells proliferate in response to peptide‐pulsed antigen‐presenting cells. Our data suggest that CD4hiCD8lo CRS thymocytes bearing the P14 tg TCR have completed positive selection and differentiate autonomously into functionally competent CD8 SP cells.
B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production
Authors: Agematsu Kazunaga , Nagumo Haruo , Yang Fen‐Chun , Nakazawa Takayuki , Fukushima Keitaro , Ito Susumu , Sugita Kanji , Mori Tetsuo , Kobata Tetsuji , Morimoto Chikao , Komiyama Atsushi
Published: March 2007

B cell immunoglobulin production is regulated by helper T cells through direct interaction and secreted cytokines. In the present study, we functionally analyzed CD27 in cord and peripheral blood B cells. Adult peripheral blood B cells were separated into CD27+ and CD27− cells, which differed in their morphology. Cord blood B cells did not express CD27, and CD27 expression on peripheral blood B cells increased with age. Only CD27+ B cells had the ability to produce immunoglobulin, which was increased by contact with a tumor necrosis factor‐related transmembrane ligand, CD70. Adult peripheral blood CD27+ B cells can be further subdivided into two discrete subtypes: IgD−CD27+ and IgD+ CD27+ B cells. IgD− CD27+ B cells produce IgG, IgM and IgA, whereas IgD+ CD27+ B cells predominantly produce IgM. The addition of activated CD4+ CD45RO T cells expressing CD70 caused down‐regulation of CD27 expression on activated B cells, and this down‐modulation was completely blocked by anti‐CD70 monoclonal antibody, indicating direct T‐B cell contact via CD27/CD70. The triggering via CD27 and CD40 additively increased the immunoglobulin production under Staphylococcus aureus Cowan strain plus interleukin‐2 stimulation. Taken together, our findings demonstrate that peripheral blood B cells are separated into subpopulations by CD27 and IgD expression and that CD27+ B cells produce large amounts of immunoglobulin by interaction with the CD70 molecule.
TAP‐translocated peptides specifically bind proteins in the endoplasmic reticulum, including gp96, protein disulfide isomerase and calreticulin
Authors: Spee Pieter , Neefjes Jacques
Published: March 2007

The endoplasmic reticulum (ER) membrane‐embedded transporter associated with antigen processing (TAP) associates with peptides in the cytosol and translocates these into the ER lumen. Here, MHC class I molecules bind a subset of these peptides and the remainder is either removed or degraded, or may be retained in the ER in association with other proteins. We have visualized peptide‐binding proteins in the ER using radioactive peptides with a photoreactive group. Besides TAP, two proteins were identified as gp96 and protein disulfide isomerase (PDI). Calreticulin, previously found in complex with TAP, only binds glycosylated peptides. In addition, two as yet unidentified, ER luminal glycoproteins (gp120 and gp170) were visualized. The effects of peptide size and sequence on binding to the ER‐resident proteins were studied by using partially degenerated peptides with photoreactive side chains. All identified proteins were able to bind peptides within the size range of peptides translocated by TAP, from 8 to more than 20 amino acids. Whereas PDI associated with all peptides tested, gp96 and gp120 showed a clear sequence preference for non‐charged amino acids at positions 2 and 9 in 9mer peptides. Thus various ER proteins, other than the MHC class I heterodimer and TAP, are able to interact with peptides albeit with a different substrate selectivity.
Trypanosoma cruzi infection does not impair major histocompatibility complex class I presentation of antigen to cytotoxic T lymphocytes
Authors: Buckner Frederick S. , Wipke Brian T. , Van Voorhis Wesley C.
Published: March 2007

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas' disease, lives free within the cytoplasm of infected host cells. This intracellular niche suggests that parasite antigens may be processed and presented on major histocompatibility complex (MHC) class I molecules for recognition by CD8+ T cells. However, the parasite persists indefinitely in the mammalian host, indicating its success at evading immune clearance. It has been shown that T. cruzi interferes with processing and presentation of antigenic peptides in the MHC class II pathway. This investigation sought to determine whether interference in MHC class I processing and presentation occurs with T. cruzi infection. Surface expression of MHC class I molecules was found to be unaffected or up‐regulated by T. cruzi infection in vitro. A model system employing a β‐galactosidase (β‐gal)‐specific murine cytotoxic T lymphocyte (CTL) line (0805B) showed: (i) in vitro infection of mouse peritoneal macrophages or J774 cells with T. cruzi did not inhibit MHC class I presentation of exogenous peptide (a nine‐amino acid epitope of β‐gal) to the CTL line, (ii) in vitro infection of a β‐gal‐expressing 3T3 cell line (LZEJ) with T. cruzi did not inhibit MHC class I presentation of the endogenous protein to the CTL line and (iii) mouse renal adenocarcinoma cells infected with T. cruzi and subsequently infected with adenovirus expressing β‐gal were able to present antigen to the β‐gal‐specific CTL line. These findings indicate that the failure of the immune response to clear T. cruzi does not result from global interference by the parasite with MHC class I processing and presentation. Parasites engineered to express β‐gal were unable to sensitize infected antigen‐presenting cells in vitro to lysis by the CTL 0805B line. This was probably due to the intracellular localization of the β‐gal within the parasite and its inaccessibility to the host cell cytoplasm.
Impaired spontaneous endocytosis of HLA‐G
Authors: Davis Daniel M. , Reyburn Hugh T. , Pazmany Laszlo , Chiu Isaac , Mandelboim Ofer , Strominger Jack L.
Published: March 2007

HLA‐G is a class Ib (non‐classical) major histocompatibility complex (MHC) protein expressed at the maternal‐fetal interface that inhibits natural killer (NK) cell‐mediated lysis in an allotype‐independent manner. Here we report that the spontaneous endocytosis of HLA‐G is severely reduced because of its short cytoplasmic tail. Class I (classical) MHC proteins on the surface of B cell trans‐fectants detected by primary and secondary antibodies underwent endocytosis at a moderate rate, whereas HLA‐G, chimeric proteins consisting of the extracellular domains of HLA‐C with the C‐terminal sequence of HLA‐G, or glycophosphatidylinositol‐tailed HLA‐C proteins, were not efficiently internalized. In addition, a mutant of β2‐microglobulin (Ser88Cys) that could be specifically labeled with Texas red (or other fluorescent probes) and exchanged into class I or class Ib MHC proteins was employed to study spontaneous internalization of MHC proteins by a non‐perturbative method independent of an antibody ligand. These data are discussed in terms of both the role of HLA‐G expressed on the fetal trophoblast and the function of the cytoplasmic tail in class I MHC proteins.
Major histocompatibility complex class I molecules interact with both subunits of the transporter associated with antigen processing, TAP1 and TAP2
Authors: Powis Simon J.
Published: March 2007

Prior to loading antigenic peptides, assembled major histocompatibility complex (MHC) class I molecules associate with the transporter associated with antigen processing (TAP) in a complex which also includes calreticulin and a recently described component, tapasin. The interaction of MHC class I molecules has been characterized as occurring exclusively with the TAP1 chain of the TAP heterodimer. In contrast, as described here, in the TAP‐deficient human cell line T2, MHC class I molecules interact with a transfected rat TAP2 polypeptide in addition to rat TAP1. Furthermore, this interaction with TAP2 also involves calreticulin and tapasin. An association with both TAP polypeptides would presumably further enhance the efficiency of peptide loading of MHC class I molecules by allowing more than one MHC class I allele proximity to the site of peptide supply on each TAP complex.
A role for the VLA‐4 integrin in the activation of human memory B cells
Authors: Silvy Anne , Altevogt Peter , Mondière Paul , Bella Chantal , Defrance Thierry
Published: March 2007

It is generally recognized that activation through membrane effector molecules such as CD40 or the B cell receptor (BCR) is mandatory to allow B cells to proliferate and differentiate into antibody (Ab)‐secreting cells in response to cytokines. We show here that purified tonsillar B cells can be stimulated directly by a cytokine combination to proliferate and secrete immunoglobulins when cultures are performed at high cell density. The contact‐mediated activation of B cells in this experimental system is strongly inhibited both by anti‐very late antigen (VLA)‐4 monoclonal Ab and by a peptide containing the LDV sequence specifically recognized by the α4 integrin binding site. These reagents also significantly suppressed the B cell responses elicited by engagement of the BCR or CD40. Our data reveal that memory B cells but not virgin or germinal center B cells are sensitive to the direct stimulatory effect of cytokines in high‐density cultures. Finally, we found that the dual expression of the α and β chains of VLA‐4 is a distinctive feature of the memory B cell population. Collectively, our findings support the notion that VLA‐4‐dependent homotypic B cell interactions can mediate a co‐stimulatory signal to human memory B cells and might participate in the B cell activation triggered through the BCR and CD40.