bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopeptide immunization without adjuvant induces potent and long‐lasting B, T helper, and cytotoxic T lymphocyte responses against a malaria liver stage antigen in mice and chimpanzees
Authors: Benmohamed Lbachir , Gras‐Masse Héléne , Tartar André , Daubersies Pierre , Brahimi Karima , Bossus Marc , Thomas Alan , Druilhe Pierre
Published: March 2007

We have employed a 26‐amino‐acid synthetic peptide based on Plasmodium falciparum liver stage antigen‐3 to evaluate improvements in immunogenicity mediated by the inclusion of a simple lipid‐conjugated amino acid during peptide synthesis. Comparative immunization by the peptide in Freund's adjuvant or by the lipopeptide in saline shows that the addition of a palmitoyl chain can dramatically increase T helper (Th) cell responses in a wide range of major histocompatibility complex (MHC) class II haplotypes, to the extent that responses were induced in mice otherwise unable to respond to the non‐modified peptide injected with Freund's adjuvant, and that the increased immunogenicity of the lipopeptide led to high and longer lasting antibody production (studied up to 8 months). B and T cell responses induced by the lipopeptide were reactive with native parasite protein epitopes, and a lipopeptide longer than ten amino acids was endogenously processed to associate with MHC class I and elicit cytotoxic T lymphocyte (CTL) responses. Finally, the lipopeptide was safe and highly immunogenic in chimpanzees, whose immune system is very similar to that of humans. Our results suggest that relatively large synthetic peptides, carefully chosen from pertinent areas of proteins and incorporating a simple palmitoyllysine, can induce not only CTL, but also strong Th and antibody responses in genetically diverse populations. Lipopeptides engineered in this way are simple to produce and purify under GMP conditions, they are well tolerated by apes, and with the enhanced immunogenicity without the need for adjuvant that we report here, they offer a quick and relatively low‐cost route to provide material for human malaria vaccination trials.
Magnitude of protein tyrosine phosphorylation‐linked signals determines growth versus death of thymic T lymphocytes
Authors: Akhand Anwarul A. , Pu Meiyi , Du Jun , Kato Masashi , Suzuki Haruhiko , Hamaguchi Michinari , Nakashima Izumi
Published: March 2007

Using concanavalin A (Con A) as a multireceptor‐reactive agonist, we studied the relationship between the growth or death of thymic T lymphocytes and the agonist concentration‐dependent magnitude of the intracellulary delivered signal. Both immature and mature thymic T lymphocytes were subjected to a high concentration of Con A‐mediated signal for apoptotic cell death. In this model, a number of cellular proteins including mitogen activated protein kinases were phosphorylated at tyrosine depending on the concentration of Con A. This effect was followed by corresponding increase in serine 73 phosphorylation of c‐jun and transcription of c‐fos. DNA fragmentation and cell membrane disruption developed concomitantly after stimulation with high concentrations of Con A. The addition of inhibitors of protein kinases which completely inhibited the growth of cells stimulated with low concentrations of Con A only partially prevented death, and even promoted DNA fragmentation of cells stimulated with high concentrations of Con A. The dissociated sensitivities of Con A‐mediated cell growth and cell death to the inhibitors were, however, shown to be due to the different efficiency of inhibition of high and low levels of intracellularly delivered signals. The results indicate that the magnitude of signaling could be the principal element that determines the growth versus death of thymic T lymphocytes.
Partial purification and characterization of a tumor necrosis factor‐α converting activity
Authors: Robache‐Gallea Sylvie , Bruneau Jean‐Michel , Robbe Hugue , Morand Valérie , Capdevila CÉCile , Bhatnagar Neerja , Chouaib Salem , Roman‐Roman Sergio
Published: March 2007

Tumor necrosis factor (TNF)‐α is initially synthesized as an extracellular membrane‐associated 26‐kDa protein that is further cleaved at Ala76‐Val77 to yield the soluble 17‐kDa form. Recently, peptide‐hydroxamate metallopro‐teinase inhibitors have been reported to block the proteolytic processing of TNF‐α, thus suggesting that the putative TNF‐α converting enzyme (TACE) is a zinc‐dependent metalloendopeptidase. In this report, we characterize a TNF‐α converting activity (TACA) that cleaves in vitro the human 26‐kDa TNF‐α at the physiological processing site. The chromatography steps followed for purification and the use of a panel of proteinase inhibitors indicate that the enzyme responsible for TACA is a membrane glycosylated metalloendopeptidase which is most likely different from the matrix‐degrading metalloproteinases. The failure of TACA to process a Val77→Gly77 precursor mutant emphasizes the importance of hydrophobic residue at P1' position. In addition, TACA is not able to cleave the mouse pro‐TNF‐α and does not catalyze in vitro the processing of other transmembrane proteins susceptible to metalloproteinase‐mediated shedding, such as interleukin‐6 or TNF receptors. These studies suggest the existence of an enzyme specific for TNF‐α within the metalloproteinases involved in the processing/shedding of a number of cytokines and cytokine receptors.
P‐selectin glycoprotein ligand‐1 mediates rolling of mouse bone marrow‐derived mast cells on P‐selectin but not efficiently on E‐selectin
Authors: Steegmaier Martin , Blanks James E. , Borges Eric , Vestweber Dietmar
Published: March 2007

It has been shown recently that mast cells play an essential role as a source of tumor necrosis factor‐α production during neutrophil recruitment to sites of bacterial infection. Increased numbers of mast cells are indeed noted at sites of wound healing and inflammation. These cells are either recruited from the bone marrow or proliferate locally under cytokine stimulation. Little is known about how mast cell progenitors extravasate into tissue. Using antibody‐like fusion proteins of mouse E‐selectin and P‐selectin, we have analyzed the ability of immature mouse bone marrow‐derived mast cells (BMMC) to interact with the endothelial selectins. The P‐selectin glycoprotein ligand‐1 (PSGL‐1) was affinity‐isolated from detergent extracts of surface biotinylated BMMC with both selectin‐IgG fusion proteins. However, only P‐selectin‐IgG, but not E‐selectin‐IgG showed significant interaction with intact BMMC as tested by flow cytometry and cell attachment assays with the immobilized fusion proteins under flow and non‐flow conditions at physiological shear stress. Thus, in spite of carrying the necessary carbohydrate modifications which enable solubilized PSGL‐1 to bind avidly to E‐selectin, PSGL‐1 on the surface of BMMC is presented in a way that prevents it from interacting efficiently with E‐selectin. Affinity‐purified rabbit antibodies against mouse PSGL‐1 almost completely blocked the interaction of BMMC with P‐selectin‐IgG in flow cytometry as well as in cell adhesion assays under static and under flow conditions. Our data reveal that PSGL‐1 is the major binding site for P‐selectin on mouse BMMC progenitors, but does not support efficient interactions with E‐selectin.
The T/B cell interaction involved in induction of the mouse IgG2ah suppression is restricted by major histocompatibility complex class I, but not class II molecules
Authors: Majlessi Laleh , Bordenave Guy
Published: March 2007

To determine the major histocompatibility complex (MHC) restriction of the T/B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell‐induced IgG2ab suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igha haplotype, when neonatally transferred into histocompatible Igha/b heterozygotes, are able to induce a specific and total suppression of the IgG2ab allotype. Nevertheless, only transfer of IgG2ab‐primed Igha T splenocytes induces this suppression in Ighb/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8+ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4+CD8+ T cell cooperation and operated via the recognition by the involved TCR of Cγ2ab‐derived peptides presented by the target B cells in an MHC haplotype‐restricted manner. Here, by using Ighb mice genetically deficient for MHC class I (β2‐microglobulin%, or β2m%) or class II (I‐Aβ%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I‐, but not class II‐restricted interaction. Indeed, the anti‐IgG2ab T cells transferred into Ighb H‐2b I‐Aβ% mice carry out the suppression process normally, while in Ighb H‐2b β2m% recipients, their suppression induction capacity is significantly inhibited. Moreover, the Cγ2ab 103–118 peptide, identified as the sole Cγ2ab‐derived peptide able to amplify the anti‐IgG2ab T cell reactivity in Igha H‐2b mice, is also able to stabilize the H‐2Db, but not the H‐2Kb class I molecules at the surface of RMA‐S (TAP2−, H‐2b) cells. These results indicate that, despite the CD4+/CD8+ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2ab+ B cells for suppression establishment.
Age‐related impaired affinity maturation and differential D‐JH gene usage in human VH6‐expressing B lymphocytes from healthy individuals
Authors: van Dijk‐Härd Iris , Söderström Ingegerd , Feld Sari , Holmberg Dan , Lundkvist Inger
Published: March 2007

To elucidate the basic molecular events underlying humoral immunity during ontogeny and senescence, we analyzed a panel of 179 polymerase chain reaction‐derived VH6‐D‐JH rearrangements from cord blood, peripheral blood, and spleen. Nucleotide sequence analysis of the CDR3 region shows that there is a difference in D and JH gene usage in functional rearrangements between lymphocytes from peripheral blood and spleen. Analysis of the VH6 gene shows that the mutational frequencies rise from 0.81% in cord blood to 1.96% in peripheral blood lymphocytes derived from young adults, and decrease to 0.80% in samples from individuals older than 50 years. The number of rearrangements carrying mutations follows a similar pattern: 22% in cord blood, 73% in the age group 20–49 years, and 57% in the age group over 50 years. The mutational frequencies among the mutated genes are, however, similar for cord blood and young adults, 2.76% and 2.51%, respectively, and 1.3% in older adults. These data show an age‐related impaired affinity maturation which might relate to the decrease in immunological responsiveness among the elderly.
Differential expression of binding sites for chemokine RANTES on human T lymphocytes
Authors: Utsunomiya Iku , Tani Kenji , Gong Wanghua , Oppenheim Joost J. , Wang Ji Ming
Published: March 2007

The C‐C chemokine RANTES, a T lymphocyte chemoattractant, is considered an important mediator of inflammation, allergy, and host defense against HIV‐1 infection. In this study, we investigated the modulation of binding of RANTES to T lymphocytes. Human peripheral blood CD3+ T cells, when freshly isolated from buffy‐coat blood, expressed a considerable number of high‐affinity binding sites for RANTES. These cells also showed significant chemotactic migration in response to RANTES in vitro. After 6–15 h incubation at 37°C, the binding of RANTES, but not of macrophage inflammatory protein‐1α (MIP‐1α) or of monocyte chemotactic protein‐3 (MCP‐3), consistently increased. Scatchard analyses indicated that the number of binding sites for RANTES increased about threefold by 15 h without any change in the affinity. The increase in RANTES binding was no longer detected by 24 h. This increase in the specific binding was mainly attributable to CD4+ T cells and was not associated with increased chemotactic activity of these cells in response to RANTES. Incubation with anti‐CD3 antibody for 15 h markedly reduced the binding capability of T cells for RANTES and was associated with decreased chemotactic activity. On the other hand, when T cells were incubated with interleukin‐2 (IL‐2) for 1 week, the specific binding for all three C‐C chemokines, RANTES, MIP‐1α, and MCP‐3 was markedly increased in comparison to cells cultured in the absence of IL‐2. These results suggest that the expression of binding sites on T cells for RANTES is differentially modulated, indicating the existence of novel receptors for RANTES that do not bind MIP‐1α.
Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM‐1
Authors: Lalor Patricia F. , Clements John M. , Pigott Rod , Humphries Martin J. , Spragg Julia H. , Nash Gerard B.
Published: March 2007

We investigated the ability of purified vascular cell adhesion molecule‐1 (VCAM‐1), adsorbed on plastic, to capture and immobilize flowing lymphocytes, and the dependence of adhesive behavior on activation of the counter‐receptor, α4β1 integrin. This integrin/immunoglobulin interaction bound lymphocytes at a wall shear stress at which the β2‐integrin family has previously been found ineffective (> 0.1 Pa), and whereas lymphocytes rolled on lower concentrations of VCAM‐1 (10 μg/ml), they were stationary at high concentrations (100 μg/ml). Activation of α4β1 integrin by Mn2+ or by antibody 12G10 or treatment of lymphocytes with phorbol ester caused transformation to stationary adhesion, and increased binding significantly only at the lower concentrations of VCAM‐1. We thus hypothesized that formation of a high density of ligand between VCAM‐1 and α4β1 integrin actively transformed lymphocyte behavior. This concept was supported by the finding that the proportion of lymphocytes rolling on the higher concentrations of VCAM‐1 increased if cells were pretreated with azide to block energy‐dependent responses, or if intracellular Ca2+ was chelated. However, not all activation responses were equivalent: only phorbol ester induced marked spreading of immobilized cells, and if pretreatment was prolonged, this agent even reduced the efficiency of initial attachment of flowing lymphocytes. Azide treatment had no effect on transformation to stationary adhesion caused by Mn2+ or activating antibody. Thus, different forms of lymphocyte activation were identifiable: external modification of integrin converted rolling to stationary attachment, did not require ATP, and was reversible; high‐density ligand binding induced an energy‐dependent signal for conversion from rolling to stationary attachment, but not spreading; and protein kinase C activation promoted stationary attachment and spreading, but not necessarily capture. VCAM‐1 is thus a versatile adhesion receptor capable of supporting all stages of leukocyte attachment, i.e. rolling, stationary, and spreading, and of ligand‐induced transformation of adhesion, although an additional signal appears necessary to promote lymphocyte spreading and migration.
T cell receptor α‐chain tail is required for protein kinase C‐mediated down‐regulation, but not for signaling
Authors: Thomas Bäckström B. , Rubin Bent , Peter Annick , Tiefenthaler Georg , Palmer Ed
Published: March 2007

Antigen stimulation through the T cell receptor (TCR) induces phosphorylation of the associated CD3 γδσ‐ and ζ‐chain cytoplasmic tails. These events lead to the induction of the intracellular signaling pathways with concomitant receptor down‐regulation. The TCR is down‐regulated from the cell surface by the activation of protein kinase C (PKC) and subsequent serine phosphorylation of the CD3 γ‐chain. We report here that the TCR α‐chain cytoplasmic tail is also necessary for PKC‐mediated internalization of the TCR complex. The requirement for the TCR α‐chain cytoplasmic tail is specific for internalization of the TCR complex, since down‐regulation of CD4 is still intact in hybridoma cells expressing a tailless TCR α‐chain. The absence of TCR internalization directly correlates with defective PKC‐mediated phosphorylation of the CD3 γ‐chain. Despite deficient PKC‐mediated TCR down‐regulation, the tailless αβ TCR still transduces antigenic signals resulting in the production of interleukin‐2. Although the TCR tails are not obviously required for signal transduction, the TCR α‐tail may serve as a targeting domain for PKC‐mediated down‐regulation of the TCR complex.
Late events in the intracellular sorting of major histocompatibility complex class II molecules are regulated by the 80‐82 segment of the class II β chain
Authors: Tan L. J. , Ceman Stephanie , Chervonsky Alexander , Rodriguez‐Paris Juan , Steck Theodore L. , Sant Andrea J.
Published: March 2007

The molecular mechanisms that regulate sorting of major histocompatibility complex (MHC) class II molecules into the endocytic pathway are poorly understood. For many proteins, access to endosomal compartments is regulated by cytosolically expressed sequences. We present evidence that a sequence in the lumenal domain of the MHC class II molecule regulates a very late event in class II biogenesis. Class II molecules containing single amino acid changes in the highly conserved 80–82 region of the β chain were introduced into invariant chain (Ii)‐negative fibroblasts with wild‐type α chain, and the derived transfectants were analyzed biochemically. Using an endosomal isolation technique, we have quantified the level of class II molecules expressed in endocytic compartments and found that in the absence of Ii, approximately 15% of total cellular class II molecules can be isolated from endosomal compartments. Mutation at position 80 enhances this localization, while changes at positions 81 and 82 ablate class II expression in endosomal compartments. In addition, we have evaluated whether the induced changes in intracellular distribution of class II molecules were due to alterations in early biosynthetic events, indicative of misfolding of the molecules, or to modulation of later trafficking events more likely to be a consequence of the modulation of a specific transport event. Despite the dramatic effects on endosomal localization induced by the mutations, early bio‐synthetic events and maturation of class II were unaffected by the mutations. Collectively, our data argue that late trafficking events that control the ability of the class II molecule to access antigens is regulated by the 80–82 segment of the MHC class II β chains.
Clustering the adhesion molecules VLA‐4 (CD49d/CD29) in Jurkat T cells or VCAM‐1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization
Authors: Ricard Isabelle , Payet Marcel D. , Dupuis Gilles
Published: March 2007

Ligation of very late antigen (VLA)‐4 (α4β1 integrin) with a cross‐linked anti‐α4 subunit monoclonal antibody (mAb) triggered a biphasic Ca2+ response in Jurkat cell populations and in peripheral human lymphocytes. Cross‐linking vascular cell adhesion molecule (VCAM)‐1 (the counter‐receptor of VLA‐4) in ECV 304 endothelial cells generated a biphasic Ca2+ response. Tumor necrosis factor‐α‐primed human umbilical cord vascular endothelial cells also responded to the cross‐linked mAb with a biphasic Ca2+ profile. Ligated VLA‐4 (Jurkat cells) or VCAM‐1 (ECV 304) stimulated the production of myo‐inositol 1,4,5‐trisphosphate. ECV 304 cells induced a biphasic Ca2+ response in Fura2‐loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2‐loaded ECV 304 cells. The Ca2+ responses in these experiments involved VLA‐4/VCAM‐1 interactions since they were significantly reduced (∼ 80%) by prior treatment of the target cells with the relevant noncross‐linked mAb. Close contact between the cells triggered mutual Ca2+ signaling as shown by spectrofluorimetric and confocal microscopy time‐dependent recordings. Fibronectin and its CS‐1 fragment (V25) triggered a sustained Ca2+ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA‐4 and VCAM‐1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca2+.
Single‐cell analyses of CD4+ T cells from αβ T cell receptor‐transgenic mice: a distinct mucosal cytokine phenotype in the absence of transgene‐specific antigen
Authors: Saparov Arman , Elson Charles O. , Devore‐Carter Denise , Bucy R. Pat , Weaver Casey T.
Published: March 2007

Development of distinct CD4+ T cell cytokine phenotypes may be conditioned by the anatomic site in which activation occurs. A double‐label in situ hybridization technique was used to characterize co‐expression of cytokine mRNA in antigen‐specific responses of Peyer's patch (PP), lamina propria (LP), and splenic (SP) CD4+ T cells isolated from αβ T cell receptor‐transgenic mice. Interleukin (IL)‐2 was the dominant cytokine expressed by antigen‐stimulated PP and SP populations, though it was expressed by a minority of the activated T cells. Cells that expressed interferon (IFN)‐γ were less frequent, and IL‐4, IL‐5, and IL‐10 were infrequent. In contrast, cells that expressed IFN‐γ or IL‐10 were most frequent in the LP population, with lower frequencies of IL‐2, and few IL‐4‐ and IL‐5‐positive cells. Co‐expression of two cytokines by the same cell was the exception, regardless of the anatomic site from which the T cells were isolated. The surface phenotype of transgene‐positive T cells isolated from each anatomic site was distinct, despite the absence of in vivo exposure to antigen for which the transgenic T cell receptor is specific. These data suggest that the cytokine responses of CD4+ T cells may be conditioned by the microenvironment, independently of specific antigen, and that the LP CD4+ T population has a distinct cytokine expression pattern with counter‐regulatory properties that may be important for homeostasis in mucosal immune tissues.
An example of idiotypic mimicry
Authors: Grandien Alf , Andersson Jan , Portnoi Denis , Coutinho Antonio
Published: March 2007

We have evaluated the impact of transgenic immunoglobulin (TGIg) expression on endogenous antibody repertoires. The transgenic system was chosen as to allow for normal recombination of endogenous Ig genes, secretion of TGIg from early development on, and distinguishing the TGIg from endogenous Ig by several serological markers on the C and V regions of the molecules. The transgenic construct encodes a complete anti‐(4‐hydroxy‐3‐iodo‐5‐nitrophenyl)acetyl (NP) antibody molecule carrying a well‐defined idiotype, bearing a λ1 light chain and a chimeric heavy chain encoded by a human α2 C region devoid of its membrane exon, and the murine B1.8 VDJ‐region. Endogenous antibody repertoires were analyzed in mitogen‐driven limiting dilution cultures, in single‐cell assays for naturally activated Ig‐secreting cells, and in hybridomas derived by direct fusion of spleen cells from unmanipulated animals. The results show that a very high frequency of splenic resting B cells and plasma cells in transgenic animals produce IgM with B1.8‐cross‐reactive idiotypes. This was confirmed by hybridoma analysis which also established that the levels of transgene expression and of idiotype‐positive IgM production by the same cell are not correlated. The affinities of idiotype‐positive endogenous Ig varied, but were generally several orders of magnitude lower than the transgene‐encoded idiotype. V regions from idiotype‐cross‐reactive IgM heavy chains showed marked diversity in sequences that were all different from the transgenic B1.8. These results are compatible with idiotypic mimicry resulting from intercellular selection based on degenerate, whole V region reactivities.
Molecular cloning of human RP105
Authors: Fugier‐Vivier Isabelle , de Bouteiller Odette , Guret Christiane , Fossiez François , Banchereau Jacques , Mattei Marie‐Geneviève , Aït‐Yahia Smina , Garcia Eric , Lebecque Serge , Liu Yong‐Jun
Published: March 2007

RP105 is a 105‐kDa type I membrane protein of the leucine‐rich repeat (LRR) family. Anti‐RP105 sensitizes B cells to antigen‐receptor‐mediated apoptosis, but protects B cells from radiation‐induced apoptosis and stimulates B cell proliferation. The sequence of the mouse RP105 has been reported. Here, we report the characterization of the human RP105. The 2.6‐kb cDNA encodes a protein of 661 amino acids which displays 78% homology with mouse RP105. The 22 LRR and the 9 potential N‐linked glycosylation sites within the extracellular region are conserved. While previous studies have shown that RP105 is expressed on surface IgM+IgD++ B cells in mice, human RP105 was shown to be expressed on all subsets of mature B cells and dendritic cells. Human RP105 gene was mapped to the long arm of chromosome 5, where numerous cytokines and receptors have been localized.
HLA‐G recognition by human natural killer cells. Involvement of CD94 both as inhibitory and as activating receptor complex
Authors: Pende Daniela , Sivori Simona , Accame Laura , Pareti Laura , Falco Michela , Geraghty Dan , Le Bouteiller Philippe , Moretta Lorenzo , Moretta Alessandro
Published: March 2007

The lack of classical human histocompatibility leukocyte antigen (HLA) molecules in human placenta prevents the recognition and lysis by maternal T lymphocytes but poses the problem of susceptibility to natural killer (NK) cell‐mediated lysis. The nonclassical HLA class I molecule HLA‐G may mediate protection from NK cells. NK cells are known to express a number of HLA class I‐specific inhibitory receptors. These include members of the immunoglobulin (Ig) superfamily (p58, p70, p140), characterized by a defined allele specificity, and CD94/NKG2A with a broad specificity for different HLA class I molecules. We analyzed a series of NK cell clones derived from normal peripheral blood expressing different NK receptors (NKR). Clones were analyzed for their cytolytic activity against the HLA class I‐negative 221 cell line either untransfected or transfected with HLA‐G (221/G) or other informative alleles, as control. All clones expressing CD94/NKG2A [as identified by the Z199 monoclonal antibody (mAb)] displayed a markedly reduced cytolytic activity against 221/G. Moreover, mAb directed to the CD94/NKG2A complex completely restored target cell lysis. Among NKG2A‐negative NK clones, different functional patterns could be detected. Clones expressing inhibitory receptors belonging to the Ig superfamily lysed 221/G target cells with equal or higher efficiency than untransfected 221 cells. These data indicated that p58, p70 and p140 do not function as HLA‐G‐specific inhibitory NKR, and that HLA‐G‐specific activating NKR also exist. Further analysis indicated that in these clones (characterized by the CD94+/NKG2A− phenotype) mAb specific for CD94, but not for the other NKR, reversed the activating effect. Infrequent clones were also isolated that, in spite of the lack of CD94/NKG2A, displayed HLA‐G specificity, thus suggesting the existence of a different, still unknown NKR.
Reduced intracellular oxidative metabolism promotes firm adhesion of human polymorphonuclear leukocytes to vascular endothelium under flow conditions
Authors: Montoya María C. , Luscinskas Francis W. , del Pozo Miguel A. , Aragonés Julían , de Landázuri Manuel O.
Published: March 2007

The interaction of polymorphonuclear leukocytes (PMN) with the vascular endothelium and their subsequent extravasation to the tissues is a key step during different physiological and pathological processes. In certain of these pathologies the oxygen tension becomes very low, leading to reduced cellular oxidative status. To evaluate the effect of lowering the intracellular redox status in the interaction of PMN with the endothelium, exposure to hypoxic conditions as well as treatment with different antioxidant agents was carried out. PMN exposure to hypoxia enhanced β2 integrin‐dependent adhesion to intercellular adhesion molecule‐1‐coated surfaces, concomitant with a decrease in the intracellular redox status of the cell. As occurs with hypoxia, treatment with antioxidants produced a decrease in the oxidation state of PMN. These agents enhanced adhesion of PMN to human umbilical vein endothelial cells stimulated with tumor necrosis factor‐α (TNF‐α), and this effect was also mediated by β2 integrins LFA‐1 and Mac‐1. Adhesion studies under defined laminar flow conditions showed that the antioxidant treatment induced an enhanced adhesion mediated by β2 integrins with a decrease in the fraction of PMN rolling on TNF‐α‐activated endothelial cells. The up‐regulated PMN adhesion was correlated to an increase in the expression and activation of integrin Mac‐1, without loss of L‐selectin surface expression. Altogether, these results demonstrate that a reduction in the intracellular oxidative state produces an enhanced β2 integrin‐dependent adhesion of PMN to stimulated endothelial cells under conditions of flow.
CD28 is not required for rejection of unmanipulated syngeneic and autologous tumors
Authors: Wen Tao , Kono Koji , Shahinian Arda , Kiessling Rolf , Mak Tak W. , Klein George
Published: March 2007

To gain a better understanding of the requirement of CD28 co‐stimulation in different types of T cell‐dependent tumor rejection responses, we performed a series of syngeneic and autologous tumor rejection experiments on CD28 knockout mice. In a preimmunization‐challenge model, virally‐induced ALC lymphoma and methylcholanthrene‐induced MC57X fibrosarcoma transplants were rejected similarly by syngeneic CD28 knockout and immunocompetent controls. ALC‐specific cytotoxic T lymphocytes (CTL) and MC57X‐specific tumor necrosis factor (TNF) release were induced in CD28 knockouts, although at a reduced level in the latter case. Secondly, the spontaneous regression of Moloney murine sarcoma virus (MMSV)‐induced primary tumors in the autologous hosts occurred equally in CD28 knockouts and in immunocompetent control mice. A comparable virus‐specific CTL response was generated in both, as revealed in cytolytic assays against RBL‐5 targets. Thirdly, the spontaneous rejection of the B7‐transfected EL‐4 lymphoma by immunocompetent hosts was abrogated in CD28 knockout mice, since more than 82% CD28 knockouts developed tumors after inoculation with B7‐transfected EL‐4 cells. Our results therefore show that CD28 co‐stimulatory molecules are not required for the rejection of unmanipulated syngeneic tumors in hyperimmunized hosts and the regression of MMSV‐induced sarcoma in autochthonous hosts.
Lymphotoxin but not tumor necrosis factor functions to maintain splenic architecture and humoral responsiveness in adult mice
Authors: Mackay Fabienne , Majeau Gerard R. , Lawton Pornsri , Hochman Paula S. , Browning Jeffrey L.
Published: March 2007

To compare the function of the tumor necrosis factor (TNF) and lymphotoxin (LT)α/β systems in the mature immune system, these two pathways were blocked with soluble receptor‐immunoglobulin (R‐Ig) fusion proteins in normal adult mice. Inhibition of LTα/β signaling using LTβR‐Ig or a blocking monoclonal antibody against murine LTβ had profound effects. The spleen lacked discrete B cell follicles and the marginal zone was altered. Less marked changes were detected in lymph nodes. LTα/β inhibition also prevented germinal center formation in the spleen and impaired Ig production in response to sheep red blood cells (SRBC) immunization. These results show that the LTα/β system is required for the maintenance of splenic architecture and normal immune responses, and not simply for the development of peripheral immune organs during ontogeny. In contrast, inhibition of the TNF/LTα pathway with TNF‐R55‐Ig did not affect the splenic architecture or the anti‐SRBC response. Splenic defects and impaired antibody responses are seen in TNF‐deficient mice, suggesting that TNF is important during development. Therefore relative to TNF, the LT system has the dominant influence on splenic organization and anti‐SRBC Ig formation in the adult mouse.
Positive and negative thymic selection in T cell receptor‐transgenic mice correlate with Nur77 mRNA expression
Authors: Xue Yintong , Chomez Patrick , Castanos‐Velez Esmeralda , Biberfeld Peter , Perlmann Thomas , Jondal Mikael
Published: March 2007

The orphan nuclear receptor Nur77 has been implicated in thymic negative selection. We studied the effect of two T cell receptor (TCR) transgenes on positive selction and Nur77 mRNA expression in thymus. DO11.10 mice, expressing a transgenic TCR specific for an ovalbumin (OVA) 323–339 peptide presented by I‐Ad, were found to have an enlarged thymus with a reduced apoptotic activity, measured by flow cytometry, reduced mitochondrial membrane potential and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) techniques. In contrast, in F5 mice expressing a transgenic TCR recognizing the influenza virus nucleoprotein (NP) 366–374 peptide restricted by Db, this positive selection effect was much less pronounced. Positive thymic selection in DO11.10 TCR+ mice correlated with a reduced level of Nur77 mRNA expression shown by Northern blot. F5 mice expressed levels close to those expressed by the wild type. Both transgenic mouse strains responded with extensive cortical apoptosis, and with up‐regulation of Nur77 mRNA, to injection of cognate peptides. As 9‐cis‐Retinoic acid (9‐cis‐RA) inhibits Nur77‐dependent apoptosis in T cell hybridomas in vitro, mice were pretreated with the drug to investigate a similar effect in vivo. However, the drug itself, at saturating concentrations, caused extensive apoptosis in immature CD4+/CD8+ thymocytes. The result demonstrates a correlation between Nur77 expression and thymic apoptotic activity, both during positive and negative selection events.
Location‐specific regulation of transgenic Ly49A receptors by major histocompatibility complex class I molecules
Authors: Fahlén Linda , Khoo Nelson K. S. , Daws Michael R. , Sentman Charles L.
Published: March 2007

Inhibitory receptors expressed on natural killer (NK) cells and T cells specific for major histocompatibility complex (MHC) class I are believed to prevent these cells from responding to normal self tissues. To understand the regulation and function of Ly49 receptor molecules in vivo, we used the CD2 promoter to target Ly49A expression to all thymocytes, T cells, and NK cells. In animals expressing its MHC class I ligand, H‐2Dd or H‐2Dk, there was a large decrease in the expression of Ly49A on thymocytes, peripheral T cells, and NK1.1+ cells. The extent of the down‐regulation of Ly49A was dependent on the expression of the MHC ligand for Ly49A and on the site where the cells were located. The level of expression of endogenous Ly49A was similarly found to be dependent upon the organ where the cells resided. Data from bone marrow chimeras indicated that most cell types may regulate Ly49A expression, but the efficacy to regulate receptor expression may vary depending on the cell type.
Two murine homologues of the human chemokine receptor CXCR4 mediating stromal cell‐derived factor 1α activation of Gi2 are differentially expressed in vivo
Authors: Moepps Barbara , Frodl Reinhard , Rodewald Hans‐Reimer , Baggiolini Marco , Gierschik Peter
Published: March 2007

Previous results have shown that pertussis toxin‐sensitive Gi proteins are likely to be involved in regulating the emigration of mature thymocytes from the thymus. In this study, a low stringency polymerase chain reaction (PCR) approach was used to identify Gi protein‐coupled cell surface receptors expressed in mouse thymocytes. Among the ten G protein‐coupled receptor cDNA isolated, the most prevalent cDNA encoded a polypeptide highly homologous to the human leukocyte‐expressed seven‐transmembrane‐domain receptor LESTR, also referred to as HIV entry cofactor, fusin, or CXCR4. Isolation of full‐length cDNA revealed that alternative RNA splicing produces transcripts encoding two isoforms of the murine LESTR, differing by the presence of two amino acids in the N‐terminal portion of the longer protein. Functional reconstitution of recombinant murine LESTR with recombinant heterotrimeric G proteins in baculovirus‐infected insect cells showed that both receptor variants mediate stromal cell‐derived factor 1α activation of the pertussis toxin‐sensitive G protein Gi2. Receptor subtype‐specific reverse transcriptase‐PCR analysis revealed differential expression of the two receptor mRNA in lymphoid tissues and brain, indicating that distinct functions are mediated by the two receptor isoforms in these tissues. The presence of LESTR mRNA in very early thymocytes as well as in immature (CD4+ CD8+) thymocytes suggests that both CD4 and LESTR are co‐expressed and render developing human thymocytes susceptible for HIV entry, which may affect generation of both CD4+ CD8− and CD4− CD8+ mature lineages.
B cell‐deficient μMT mice as an experimental model for Mycoplasma infections in X‐linked agammaglobulinemia
Authors: Berglöf Anna , Sandstedt Karin , Feinstein Ricardo , Bölske Göran , Smith C. I. Edvard
Published: March 2007

B cell‐deficient μMT mice were investigated as an experimental model for human X‐linked agammaglobulinemia (XLA). Mice were intranasally infected with Mycoplasma pulmonis and in 16 out of 17 μMT mice, dissemination of the bacteria from the airways was observed. More than 50% of these mice developed arthritis and/or changes in periarticular tissues. Mycoplasmal infection in μMT mice thus resembles the disease seen in XLA patients implying the usefulness of the model.
Expression of mouse CD43 in the B cell lineage of transgenic mice causes impaired immune responses to T‐independent antigens
Authors: Ostberg Julie R. , Dragone Leonard L. , Borrello Melinda A. , Phipps Richard P. , Barth Richard K. , Frelinger John G.
Published: March 2007

CD43 (leukosialin), a sialylated glycoprotein expressed on the surface of most hematopoietic cells, has been implicated in cell adhesion and signaling. However, its precise physiological function remains unclear. We used mouse CD43 (mCD43)‐immunoglobulin enhancer‐transgenic (TG) mice to study the role of mCD43 in vivo. Previous work revealed that mCD43 expression on mature B cells in these mice resulted in immunodeficiency to T‐dependent (TD) antigens (Ag), possibly by impairing B‐T cell interactions. In the present study we have immunized the TG mice with the T‐independent (TI) Ag fluorescein‐(Fl) lipopolysaccharide (LPS) (TI type 1 Ag) and Fl‐Ficoll (TI type 2 Ag). Surprisingly, the mCD43‐Ig enhancer expressing mice were impaired in their ability to mount humoral responses to both Fl‐LPS and Fl‐Ficoll, and had decreased numbers of cells responding to Ag in vivo. Flow cytometric analysis was performed on peritoneal B‐1 cells, a population which often plays a major role in humoral responses to TI Ag such as bacterial Ag. This analysis revealed similar B220, IgM and CD5 expression patterns for the TG and nontransgenic (NTG) B‐1 cells. In addition, purified peritoneal B‐1 cells from TG and NTG mice were able to respond to LPS. Stimulation of splenic B cells in vitro with Fl‐LPS and Fl‐Ficoll revealed that, in contrast to NTG B cell responses, TG B cell responses could not be enhanced by co‐culture with T cells. However, soluble T cell factor enhancement of the TG B cell responses was normal. These data suggest that the mCD43 expression on B cells may inhibit cell interactions that are important for enhanced TI Ag responses. The anti‐adhesive forces of mucins in general may thus be critical in regulating both TD and TI humoral responses.
Induction of endothelial cell surface adhesion molecules by tumor necrosis factor is blocked by protein tyrosine phosphatase inhibitors: Role of the nuclear transcription factor NF‐xB
Authors: Dhawan Subhash , Singh Sanjaya , Aggarwal Bharat B.
Published: March 2007

Recent studies from our laboratory have indicated that protein tyrosine phosphatase (PTPase) inhibitors can down‐modulate the tumor necrosis factor (TNF)‐mediated activation of the nuclear transcription factor NF‐xB in ML‐1a, a monocytic cell line (Singh and Aggarwal, J. Biol. Chem. 1995: 270: 10631). Since TNF is one of the major inducers of various adhesion molecules in human endothelial cells and their expression is known to require the activation of NF‐xB, we examined the effect of PTPase inhibitors on the TNF‐mediated induction of intracellular adhesion molecule (ICAM)‐1, vascular cell adhesion molecule (VCAM)‐1 and endothelial leukocyte adhesion molecule (ELAM)‐1. Like ML‐1a, human dermal microvessel endothelial cells (MVEC) treated with TNF rapidly activated (within 30 min) NF‐xB; this effect was completely abolished by co‐treatment with phenylarsine oxide (PAO), a specific inhibitor of PTPase. The induction of ICAM‐1, VCAM‐1, and ELAM‐1 by TNF in MVEC occurred within 6 h and was also completely down‐regulated by PAO in a dose‐dependent manner. PAO was found to be effective even when added 3 h after TNF, suggesting a rapid mode of action of this inhibitor. Besides PAO, other inhibitors of PTPase, including pervanadate and diamide, also blocked TNF‐dependent NF‐xB activation and induction of all the three adhesion proteins. Consistent with these results, the attachment of monocytes to MVEC was also blocked by the PTPase inhibitors. Thus, overall, our results demonstrate that a PTPase is involved either directly or indirectly in the pathway leading to the induction of endothelial cell adhesion molecules by TNF. Because of their role in cell adhesion, PTPase may provide a novel target of drug development for treatment of inflammation, atherogenesis, and tumor metastasis.
The migratory behavior of murine CD4+ cells of memory phenotype
Authors: Tietz Wolfgang , Hamann Alf
Published: March 2007

Lymphocyte differentiation is connected with profound alterations in the migratory pattern of lymphocytes. Whereas naive cells predominantly recirculate through lymphoid tissues, activated lymphocytes acquire an increased preference for immigration into non‐lymphoid tissues and a reduced capacity for recirculation via high endothelial venules (HEV). A variety of data had indicated that memory‐related subpopulations of cells in man and sheep, classified by the low expression of the CD45RA isotype, also lack the capacity to recirculate via HEV. However, recent data in the rat called these results into question. We therefore analyzed the migration properties of murine CD4+ T cell subpopulations defined by several markers used to distinguish memory from naive CD4+ cells in mice, namely CD45RB, L‐selectin and CD44. Our data clearly show that the majority of putative memory cells expressing either low levels of CD45RB, low levels of L‐selectin or high levels of CD44 display a strongly reduced capacity for direct entry into lymphoid tissues, including the spleen, from the blood stream. The accumulation in peripheral lymph nodes is further reduced by treatment with anti‐L‐selectin antibody, which blocks their entry via HEV. This indicates that memory CD4+ T cells are not excluded from crossing lymph node HEV, and that the numbers of cells entering the node via this route exceed the numbers entering via the afferent lymph, at least in the absence of local inflammation. Concomitantly, a strongly enhanced localization of cells of the memory phenotype is observed in lung and liver as compared with naive cells. Trafficking to specific sites such as skin or gut mucosa is not a prominent feature of the total population of memory cells. The trafficking to lung and liver and an increased ability to bind to dendritic cells, demonstrable in in vitro adhesion assays, suggest a more sessile phenotype of most memory cells. With respect to these properties, memory cells have a surprizing similarity to fully activated lymphocytes.
Opposite role for interleukin‐4 and interferon‐γ on CD30 and lymphocyte activation gene‐3 (LAG‐3) expression by activated naive T cells
Authors: Annunziato Francesco , Manetti Roberto , Cosmi Lorenzo , Galli Grazia , Heusser Christoph H. , Romagnani Sergio , Maggi Enrico
Published: March 2007

Polarized human type 1 and type 2 T helper cells not only produce different sets of cytokines, but they also preferentially express certain activation markers, such as lymphocyte activation gene‐3 (LAG‐3) and CD30, respectively. In this study we have examined the LAG‐3 and CD30 expression in relation to the lineage commitment of human naive CD4+ T cells, as assessed at the single‐cell level of committed T cells. Purified CD45RA+ umbilical cord blood T lymphocytes were activated with phytohemagglutinin and interleukin (IL)‐2 in the absence or presence of interleukin IL‐4 or IL‐12 and assessed for CD30 and LAG‐3 expression, as well as for intracellular cytokine synthesis. Significant numbers of CD30+ cells were only found in CD4+ and CD8+ T lymphocytes of cultures primed with IL‐4, which developed into cells able to produce IL‐4 and IL‐13 in addition to interferon (IFN)‐γ. By contrast, LAG‐3 expression was strongly up‐regulated in CD4+ and CD8+ T cells from cultures primed with IL‐12, which developed into high numbers of IFN‐γ producers. The addition of a neutralizing anti‐IFN‐γ antibody to IL‐12‐primed CD4+ T cell cultures virtually abolished the development of LAG‐3‐expressing CD4+ T cells. Taken together, these data suggest that CD30 expression is dependent on the presence of IL‐4, whereas LAG‐3 expression is dependent on the production of IFN‐γ during the lineage commitment of human naive T cells.
Double‐positive T cell receptorhigh thymocytes are resistant to peptide/major histocompatibility complex ligand‐induced negative selection
Authors: Ghendler Yoseph , Hussey Rebecca E. , Witte Torsten , Mizoguchi Emiko , Clayton Linda K. , Bhan Atul K. , Koyasu Shigeo , Chang Hsiu‐Chiang , Reinherz Ellis L.
Published: March 2007

To investigate negative selection events during intrathymic ontogeny, we established T cell receptor (TCR)‐transgenic mice [N15tg/RAG‐2−/− (H‐2b)] expressing a single TCR specific for vesicular stomatitis virus nuclear octapeptide N52–59 (VSV8) in the context of the major histocompatibility complex (MHC) class I molecule, Kb. Administration of VSV8 in vivo induced apoptosis in less than 4 h, deleting the majority of immature double‐positive (DP) thymocytes by 24 h. In contrast, DP TCRhigh as well as single‐positive (SP) thymocytes were refractory to this death process. Moreover, DP TCRhigh cells differentiated into SP thymocytes in vitro and in vivo, maturing into functional cytotoxic T lymphocytes upon intrathymic transfer to β RAG 2−/− recipients. Hence, negative selection processes involving MHC‐bound peptide ligands are operative only prior to the late DP thymocyte stage in this MHC class I‐restricted TCR transgene system.