bluuebunny/crossref_metadata_2025 huggingface dataset:
Transcriptional diversity at the duplicated human CD8β loci
Authors: Disanto James P. , Smith Deborah , De Bruin Derik , Lacy Elizabeth , Flomenberg Neal
Published: March 2007

In order to understand the structural organization of the human CD8β locus, genomic clones containing CD8β sequences were isolated and analyzed. Physical linkage of these clones with the CD8α locus using pulsed‐field electrophoresis revealed a duplication of the CD8β locus. CD8B‐1 lies 35 kb upstream from the CD8α locus and contains eight exons, including four alternatively spliced cytoplasmic exons. The CD8B‐2 gene contains six exons and is at present unlinked to CD8B‐1. Analysis of sequences upstream to the leader exon of the CD8B‐1 and CD8B‐2 genes revealed a GC‐rich promoter which lacks canonical “CCAAT” and “TATA” motifs, but which has sites for multiple transcriptional activators and three additional elements which are conserved in the murine CD8β promoter. Seven unique CD8β cDNA isoforms were isolated and characterized, which derive from alternative splicing of the transmembrane and/or cytoplasmic exons. Three cDNA are membrane spanning, while the remaining four isoforms lack a transmembrane region and are potentially secreted. These transcripts are differentially expressed in the thymus and in the periphery. Transaction experiments in murine fibroblasts confirmed that the membrane CD8β isoforms could be expressed as heterodimers with the CD8α chain. The regulated expression of multiple CD8β cytoplasmic isoforms and their potential role in T lymphocyte signal transduction is discussed.
Only a small proportion of splenic B cells in adults are short‐lived virgin cells
Authors: Chan Eric Y.‐T. , Maclennan Ian C. M.
Published: March 2007

A cohort of newly produced virgin B cells was followed from the marrow to the spleen of non‐immunized clean rats, which showed minimal antigen‐driven proliferation of B cells in their spleens. The progenitors of this cohort of virgin cells were labeled in vivo over 12 h with the thymidine analogue 5‐bromo‐2′‐deoxyuridine (BrdUrd) and their non proliferating progeny left the marrow 2‐3 days later. This coincided with the arrival of labeled B cells in the red pulp and T zones of the spleen. These appear to be short‐lived as few remained a week after the label was given. The short‐lived newly produced virgin B cells can only comprise a minority of splenic B cells, for it is shown that only 20% of splenic B cells are found in the red pulp and T zone. It is calculated that newly produced virgin B cells are likely to make up between 5% and 10% of splenic B cells. In the marginal zones and follicular mantle respective medians of 3.3% and 1.8% were already labeled at 1 day from the start of the BrdUrd pulse. The appearance of these cells seems likely to result from antigen‐driven B cell proliferation outside the marrow, for labeled virgin B cells have not started to leave the marrow at this stage. During day 2 and 3 the proportion of labeled follicular mantle B cells rose to 3.4%, which might in part reflect the recruitment of newly produced virgin B cells to the pool of recirculating follicular B cells. After day 3 in the follicles and day 1 in the marginal zones the proportion of labeled cells did not vary significantly through day 7. This appears to confirm the comparative longevity of the cells in these zones, which contain 80% of the non‐proliferating splenic B cells of adult rats.
Complement‐dependent synergistic effects of rat monoclonal IgG antibodies in vivo
Authors: Yousaf Nasim , Howard Jonathan C. , Williams Bryan D.
Published: March 2007

We describe studies aimed at maximizing the effector mechanisms responsible for eliminating target erythrocytes from the circulation in a fully homologous opsonization system in vivo. The effects on the subsequent fate of target erythrocytes were examined in both normal and decomplemented rats preinjected with a variety of rat IgG monoclonal antibodies (mAb) directed against different epitopes on the RTlAa, the classical class I major histocompatibiliy complex antigen of the DA rat. In general, the clearance of both DA and (DA × PVG)F1 erythrocytes in normal rats preinjected with various pairs of noncompetitive mAb was very rapid when compared with the overall clearance patterns seen with individual antibodies. With all mAb combinations containing IgG2b or IgG2a, an intact complement system was an essential requirement for augmenting the initial clearance and promoting hepatic sequestration of these target cells. The removal of (DA × PVG)F1 erythrocytes, expressing half as much antigen, was considerably slower than the DA cells for each antibody pair tested although a notable degree of heterogeneity was observed in the overall behavior of both types of target cells with different mAb combinations. Our results suggest that the limiting effects of low antigen density on the target cells combined with the use of mAb of an isotype like the rat IgG2a can be overcome using pairs of mAb that recognize different epitopes on the same target antigen.
Human interleukin‐5 induces staphylococcal A Cowan 1 strain‐activated human B cells to secrete IgM
Authors: Bertolini John N. , Sanderson Colin J. , Benson Elizabeth M.
Published: March 2007

Studies on the role of human interleukin (IL)‐5 in B cell growth and differentiation have yielded conflicting results. To clarify this issue, we studied the role of purified recombinant IL‐5 on activated human B cells which were depleted of Tcells and adherent cells. Human IL‐5 augments IgM secretion, but not IgG or IgA secretion of purified human B cells activated with staphylococcal A Cowan 1 strain (SAC). However, the period of B cell activation with SAC is critical for the B cell to respond to IL‐5. After 24 h of SAC activation, human B cells are responsive to the IL‐5 signal, but with longer periods of activation, IL‐5 responsiveness diminishes. This may explain some of the previous conflicting results. The IgM enhancement was not seen when B cells were activated with pokeweed mitogen. In addition, human recombinant IL‐4 synergized with IL‐5 in augmenting IgM secretion by SAC‐activated B cells, while IL‐5 synergized with IL‐2 to augment IgM, IgG and IgA secretion by SAC‐activated B cells. As the purified IL‐5 was derived from a COS‐1 cell supernatant, and COS‐1 cells secrete IL‐6, we examined whether a polyclonal IL‐6 antibody blocked the IgM‐enhancing activity of IL‐5. IL‐6 antibody did not block the IL‐5 enhancement of IgM secretion, but a monoclonal antibody to IL‐5 inhibited the human IL‐5 activity on human B cells. These results demonstrate that human IL‐5 augments IgM secretion of SAC‐activated human B‐cells. In addition, this lymphokine synergizes with IL‐4 and IL‐2 in supporting Ig secretion.
A sequence pattern for peptides presented to cytotoxic T lymphocytes by HLA B8 revealed by analysis of epitopes and eluted peptides
Authors: Sutton Julian , Rowland‐Jones Sarah , Rosenberg William , Nixon Douglas , Gotch Frances , Gao Xiao‐Ming , Murray Nick , Spoonas Anna , Driscoll Paul , Smith Michael , Willis Anthony , McMichael Andrew
Published: March 2007

HLA B8‐restricted cytotoxic T lymphocytes (CTL) specific for influenza A virus were generated and shown to recognize the nucleoprotein (NP). The dominant epitope was mapped using recombinant vaccinia viruses that expressed fragments of the NP and then synthetic peptides baseds on the NP amino acid sequence. The peptide 380‐393 was first identified and further refined; it was shown that the glutamic acid at position 380 was essential for recognition by CTL and that the nonamer 380‐388 was the optimum peptide. Six HLA B8‐positive influenza immune donors that we have tested respond to this peptide as part of their influenza‐specific CTL response. The amino acid sequence of the peptide epitope was compared to six other known virus peptides known to be restricted by HLA B8 and a sequence homology was identified, which predicted nonamer and octamer epitope sequences. Probable anchor residues were identified at peptide residues 3 (lysine/arginine), 5 (lysine/arginine) and 9 (leucine/isoleucine). Support for this pattern came from sequencing peptides eluted from purified HLA B8 molecules, where lysines were predominant at positions 3 and 5. One of the predicted epitope peptides was made and shown to be recognized by specific CTL. These and the two others were shown to compete with NP 380‐388 for binding to HLA B8. A model was made of the HLA B8 molecule and negatively charged pockets predicted, which could accomodate the positively charged side chains of the peptide anchor residues.
Covalent binding of guanine nucleotides to the CD3‐γ chain of the T cell receptor/CD3 complex
Authors: Peter Marcus E. , Wileman Thomas , Terhorst Cox
Published: March 2007

In a search for proteins involved in signal transduction through the T cell receptor (TcR/CD3 complex), a recently developed highly efficient method for labeling of nucleotide binding proteins in permeabilized cells was applied. Here, we report that human CD3‐γ could be labeled by periodate‐oxidized [α‐32P] GTP (GTPoxi). In contrast to GTPoxi labeling of CD3‐ξ, (Peter, M. E., Hall, C, Ruhlmann, A., Sancho, J. and Terhorst, C, EMBOJ. 1992. 11: 933), GTP‐specific labeling of CD3‐γ reached a maximum when nucleotides were added 60 min prior to the cross‐linking reaction. As CD3‐γ did not contain a known consensus sequence for nucleotide binding and since labeling kinetics of CD3‐γ coincided with those of cytosolic GTP‐binding proteins, labeling may have been caused by a GTP‐binding protein. This putative protein was not T cell specific because labeling of CD3‐γ could also be achieved when expressed in the endoplasmic reticulum of Chinese hamster ovary (CHO) cells. In CHO cells, labeling by GTPoxi took place only when CD3‐γ was associated with CD3‐ξ, whereas labeling could not be established upon association of CD3‐γ with CD3‐δ or TcR α. The observation that CD3‐γ was labeled without leaving the endoplasmic reticulum led to the hypothesis that the association of CD3‐γ with a GTP‐binding protein might be involved in an early step of the TcR/CD3 complex formation or transport.
The soluble interleukin‐6 receptor is generated by shedding
Authors: Mülberg Jürgen , Schooltink Heidi , Stoyan Tanja , Günther Monika , Graeve Lutz , Buse Gerhard , Mackiewicz Andrzej , Heinrich Peter C. , Rose‐John Stefan
Published: March 2007

The ligand‐binding subunit (gp80) of the human interleukin‐6 receptor (IL‐6R) was transiently expressed in COS‐7 cells. The metabolically labeled protein was shown to be quantitatively released from the membrane within 20 h. We identified the protein released from the transfected COS‐7 cells after purification to homogeneity and N‐terminal sequencing as a soluble form of the gp80/IL‐6R. Shedding of the gp80 protein was strongly induced by 4β‐phorbol‐12‐myristate‐13‐acetate, indicating that the process was regulated by protein kinase C (PKC). This was further corroborated by the finding that co‐transfection of a PKC expression plasmid led to enhanced shedding of the gp80 protein. Since shedding of gp80 could not be prevented by treatment of the cells with inhibitors of all known classes of proteases, a novel protease seems to be involved. As a control, an unrelated membrane protein (vesicular stomatitis virus glycoprotein) was transfected into COS‐7 cells and analyzed for shedding. Since the turnover of this protein was not mediated by shedding, we conclude that the release of gp80 from COS‐7 cells is a specific process. The shed gp80 protein specifically binds IL‐6, and this complex shows biological activity on human hepatoma cells. Human peripheral blood monocytes released a soluble form of the gp80 protein into the culture medium upon PMA treatment indicating that PKC‐regulated shedding is the physiological mechanism of generation of the soluble IL‐6R.
Evidence for a human IgG1 class switch program
Authors: Irsch Johannes , Hendriks Rudolf , Tesch Hans , Schuurman Ruud , Radbruch Andreas
Published: March 2007

In activated murine B lymphocytes, immunoglobulin class switch recombination occurs as a highly regulated process which is targeted to distinct switch regions. Here we present first evidence that in human B lymphocytes, switch recombination is targeted to distinct switch regions as well. In a panel of clonally unrelated IgG1‐expressing human B cells, immortalized by Epstein‐Barr virus (EBV) transformation, seven out of nine cells show switch recombination between Sμ and Sγ1 on both alleles, the active and inactive one. The remaining cells show no switch recombination on the inactive IgH locus. The very strong correlation of switch recombination on both alleles of IgG1‐expressing cells proves that class switch recombination to IgG1 is not random but directed in human B lymphocytes.
Diverse T cell receptor β chain usage by rat encephalitogenic T cells reactive to residues 68‐88 of myelin basic protein
Authors: Sun Deming , Le Junyi , Coleclough Christopher
Published: March 2007

Encephalitogenic T cells not only cause experimental autoimmune encephalomyelitis (EAE), but they induce resistance against subsequent induction of the disease as well. The T cell receptor (TcR) of encephalitogenic T cells is believed to contribute to their vaccinating activity. Findings in support of this assumption include the apparent restricted use of particular TcR elements, such as Vβ8.2. However, results from other laboratories including ours do not support this idea. We previously showed that rat T cells reactive against the conserved encephalitogenic epitope of myelin basic protein [MBP (87‐99)] use the TcR in a heterogeneous fashion (Sun, D. et al., Eur. J. Immunol. 1992. 22: 591). Here we show, in Lewis rats, that the TcR β chain usage of T cells specific for the dominant MBP (68‐88) epitope is not restricted to Vβ8.2. Not only did such cells rely on diverse Vβ chains, but some non‐Vβ8‐bearing cells were highly encephalitogenic. We also show that antigen‐presenting cells (APC) play an important role in determining the TcR usage of MBP‐specific T cells. Stimulation of MBP (68‐88)‐specific T cell lines by cloned APC derived from different sources resulted in selection of encephalitogenic T cells bearing different TcR β chains.
Activated T lymphocytes in the celiac lesion: Non‐proliferative activation (CD25) of CD4+ α/β cells in the lamina propria but proliferation (Ki‐67) of α/β and γ/δ cells in the epithelium
Authors: Halstensen Trond S. , Brandtzaeg Per
Published: March 2007

In order to identify any dominating subset of activated T cells in the celiac lesion, we examined CD3+, CD4+, CD8+ and T cell receptor (TcR) γ/δ+ Lymphocytes in jejunal cryosections from 25 patients with celiac disease and 10 controls by three‐color immunofluorescence staining for expression of the nuclear proliferation marker detected by monoclonal antibody (mAb) Ki‐67 and the p55 α chain of interleukin‐2 receptor (CD25). mAb Ki‐67+ intraepithelial lymphocytes (IEL) were exclusively observed in celiac patients. The median proportion of CD3+ IEL positive for Ki‐67 increased from nil in controls to 4.5% in partly treated (range 0‐19.0%; n = 10; p = 0.05) and 12.8% in untreated celiac disease (range 4.0‐30.7%; n = 15; p 0.005). Only 1.5% of CD3+ subepithelial T cells expressed the Ki‐67 marker in celiac disease (range 0‐9.5%). Two‐ and three‐color staining combining mAb to CD3 and Ki‐67 with mAb to CD4, CD8 or TcR5 showed that both TcR α/β+ CD8+ and TcR γδ+ (but not CD4+) mucosal T cells proliferated in the epithelium. By contrast, CD25 were almost exclusively expressed on CD4+ T cells in the lamina propria. The percentage of CD25+ T cells increased significantly from 1.7% in controls (range 0‐2.9%) to 7.5% in partly treated (range 0.8‐17.8%, p 0.002), and to 14.65% in untreated celiac disease (range 3.9‐21%, p 0.002). These results suggest that gluten ingestion in celiac disease induces proliferative activation of TcR α/β+ CD8+ and TcR γδ+ IEL but non‐proliferative activation (lymphokine production?) of lamina propria CD4+ T cells.
Constitutive expression of high levels of soluble mouse CD4 in transgenic mice does not interfere with their immune function
Authors: Weber Susanna , Traunecker André , Karjalainen Klaus
Published: March 2007

Interactions of CD4 with the major histocompatibility complex (MHC) class II molecules are crucial during thymic development and subsequently for the function of single‐positive CD4+CD8− T lymphocytes. Here, we have investigated the potential effects of soluble CD4 (sCD4) on the immune system. We generated two different transgenic mouse lines, which constitutively expressed either ˜100 μg/ml of monovalent or ˜20 μg/ml f decavalent mouse sCD4 molecules in their sera. Analysis of these mice revealed no differences compared to control littermates, e.g. the single‐positive CD4+ cells developed normally and these cells responded to allogeneic and anti‐CD3 antibody stimuli like the cells from control mice. Furthermore, the T helper cell function for antibody responses in vivo were not affected. Our data provide evidence that, in mouse, the CD4‐MHC class II‐interaction has very low affinity. Since sCD4 is considered to be a therapeutical agent for human immunodeficiency virus infection, these findings are not only of basic, but also of clinical interest.
Biased usage of two restricted VH gene segments in Vh replacement
Authors: Komori Toshihisa , Minami Yusaku , Sakato Nobuo , Sugiyama Haruo
Published: March 2007

AT8‐1‐12‐5, an intracytoplasmic γ2b‐producing (μ‐γ2b+) pre‐B cell line transformed with Abelson murine leukemia virus continuously generated intracytoplasmic μγproducing (μ+μ2b+ cells during propagation in culture. Southern blotting, DNA cloning and sequence analysis showed that these μ. +μ2b+ cells were generated from the μ‐ γ2b+ pre‐B cells by VH replacement events. The gene segments involved in these replacement events were restricted to only two VH gene segments, Vh7.1 (Vh7183 family) and Vh6.2 (VhQ52 family). Cell staining using the monoclonal anti‐VH141 antibody 3‐5‐6f that specifically recognized the VH6.2 but not VH7.1 gene products indicated that half of the VH replacement events occurring in AT8‐1‐12‐5 used the VH6.2 gene segment. Deletion mapping indicated that the incoming VH gene segments, VH7.1 and VH6.2 were proximal to the resident VH gene segment, VH12.5 (VH7183 family). These results provided direct evidence of the biased usage of VH gene segments in VH replacement and have several implications for the mechanisms of VH replacement.
Available λ B cell repertoire in the mouse: Evidence of positive selection by environmental factors
Authors: Nadel Bertrand , Drapier Anne‐Marie , Cazenave Pierre‐André , Sanchez Pierre
Published: March 2007

We have recently shown that, from two BALB/c mice treated with rabbit anti‐Cλ2/Cλ3 antibodies coupled to lipopolysaccharide, variable heavy chain (VH) family repertoires associated with λ2 or λ3 light chains can differ from one λ. subtype to another and from one individual mouse to another. Indeed, 4 out of 6 λ2 (VxJ2) hybridomas from one mouse preferentially expressed the VH10 family while 3 out of 8 λ2 (V2J2) and 5 out of 8 λ2 (VxJ2) hybridomas from a second mouse preferentially expressed the S107 and VGAM3.8 VH families, respectively. In this report, we describe the structural basis of such preferential pairings by sequence analysis of the 12 λ2 hybridomas. The sequence comparison of their VH regions show that each preferential association of a VH family to one Vλ region is restricted to the use of a single member or very closely related members inside a VH family and that a great variability of CDR3 of heavy chain is observed. We, therefore, suggest that environmental factors can modify the available XλB cell repertoire through a positive selection of particular VH/Vλ pairings. Moreover, our data support that this selection does not require clonal expansion and punctual somatic mutation.
Involvement of β1 integrins in the binding and entry of Trypanosoma cruzi into human macrophages
Authors: Fernández Mónica A. , Muñoz‐Femández Ma Angeles , Fresno Manuel
Published: March 2007

We have investigated the role of integrin molecules in the binding and entry of Trypanosoma cruzi into human macrophages, with the help of monoclonal antibodies (mAb). Addition of Lia 1/2 mAb and in lesser extent of Lia 1/5 mAb, which are both specific for the β1 subunit of the VLA integrin family, to human macrophages blocked T. cruzi uptake and subsequent replication inside the macrophages. This inhibition correlated with their respective ability to block Fibronectin (Fn) binding to macrophages. Furthermore, another anti‐β1 mAb, Alex 1/4, which binds to a different epitope on the β1 molecule and was unable to block Fn binding, did not affect T. cruzi invasion. The inhibition by Lia 1/2 and Lia 1/5 was dose dependent and clearly observable with doses as low as 1 μg/ml. Moreover, this inhibition was T. cruzi specific since the Lia 1/2 and Lia 1/5 were unable to block uptake of Leishmania pifanoi or Escherichia coli by human macrophages. In contrast, the TS 1/18 mAb, which blocks ligand binding to β2 integrin, inhibited entry of L. pifanoi but not of T. cruzi. Finally, mAb specific for the a4 and a5 subunits of the two major Fn binding molecules of macrophages (VLA‐4 and VLA‐5, respectively), either alone or in combination, were poor inhibitors of T. cruzi uptake, suggesting that several members of the VLA family, including VLA‐4 and VLA‐5, are involved in binding and entry of T. cruzi into macrophages.
Chronic myelogenous leukemia‐derived basophilic granulocytes express a functional active receptor for the anaphylatoxin C3a
Authors: Kretzschmar Titus , Jeromin Andreas , Gietz Claudia , Bautsch Wilfried , Klos Andreas , Köhl Jörg , Rechkemmer Gerhard , Bitter‐Suermann Dieter
Published: March 2007

The receptor for the inflammatory peptide C3a has scarcely been examined on human cells. This work demonstrates that human tumor‐derived basophilic granulocytes express C3a receptors, and presents parts of the hitherto unknown C3a‐signal transduction. When incubated with IL‐3, these cells specifically liberated histamine on C3a stimulation. Independent from IL‐3, 240000 ± 100000 receptors per cell with a Kd of 5.6 ± 0.9 nM were determined. (Ca2+)j increased from 120 ± 35 nM to 300 ± 80 nM after a C3a challenge, as measured by digital imaging fluorescence microscopy, and rested at its basal level in the presence of C3a‐desArg, the immediate catabolic product of C3a in vivo. This (Ca2+ )i increase could be completely desensitized homologously by C3a as well as inhibited by up to 75% by pertussis toxin. Thus, tumor‐derived basophils are suitable for cloning of the human C3a receptor.
Interleukin‐4 transgenic mice of resistant background are susceptible to Leishmania major infection
Authors: Leal Ludmila M. C. C. , Moss David W. , Kuhn Ralf , Miiller W. , Liew Foo Y.
Published: March 2007

The outcome of cutaneous leishmaniasis is dependent on the balance of Th1 and Th2 cells. In the murine model, Th1 cells are host‐protective whereas the Th2 cells are disease‐promoting. However, the in vivo role of interleukin‐4 (IL‐4), a signature product of Th2 cells, is uncertain. We compared the course of Leishmania major infection in the genetically resistant 129/Sv mice and the mutant 129/Sv mice transgenic for the murine IL‐4 gene under the control of the immunoglobulin heavy chain enhancer and promoter. We report here that in contrast to their wild‐type parents, the IL‐4 transgenic mice are susceptible to L. major infection. This is associated with the development of inexorably progressive lesions and parasite loads. Spleen cells from infected transgenic mice produced significantly higher levels of IL‐4 but lower amounts of interferon‐γ when stimulated in vitro with leishmanial antigens compared to those from infected normal 129/Sv mice. Furthermore, sera from the infected transgenic mice contained higher levels of IL‐4 and IgE than the sera of infected normal 129/Sv mice. These results, therefore, establish in a new animal model that IL‐4 promotes disease development in murine cutaneous leishmaniasis.
Expression of interleukin‐1 receptor antagonist (IL‐1ra) by human circulating polymorphonuclear cells
Authors: Re Fabio , Mengozzi Manuela , Muzio Marta , Dinarello Charles A. , Mantovani Alberto , Colotta Francesco
Published: March 2007

After appropriate stimulation, mononuclear phagocytes express a specific inhibitor of interleukin (IL)‐1, now re‐named IL‐1 receptor antagonist (IL‐1ra). In this study we have examined the production of IL‐1ra by polymorphonuclear cells (PMN). Human PMN isolated from peripheral blood expressed low but detectable levels of IL‐1ra transcripts, which were considerably augmented after treatment with lipopolysaccharrides (LPS) and cytokines [IL‐4, granulocyte (G)‐and granulocyte macrophage (GM)‐Colony Stimulating factor (CSF), and tumor necrosis factor (TNF)]. The levels of induced IL‐1 ra transcripts were comparable to those observed in endotoxin‐stimulated human monocytes. By contrast IL‐1β interferon (IFN)‐γ and chemotactic factors (fMLP, C5a and IL‐8) failed to promote IL‐1ra expression in PMN. IL‐1ra induction by LPS reached peak levels at 10 ng/ml after 3‐6 h and remained sustained 24 h after stimulation. Induction by LPS and GM‐CSF appears to be at the transcriptional level, as assessed by inhibiting mRN A synthesis with actinomycin D. Inhibition of protein synthesis by cycloheximide superinduced both basal and inducible IL‐1ra mRNA. In addition to expressing mRNA, PMN also produce IL‐1ra protein. Secretion of IL‐1ra was induced in PMN treated with LPS, IL‐4 and GM‐CSF, but not by IL‐1β IFN‐γ and fMLP, thus yielding results that paralleled those seen in Northern blot experiments. These data indicate that, among myelomonocytic cells, PMN, in addition to mononuclear phagocytes, can express IL‐1ra, suggesting that PMN, while exerting a series of pro‐inflammatory activities, may also modulate the inflammatory potential of IL‐1 in tissues.
Malaria vaccine: Immunization of mice with a synthetic T cell helper epitope alone leads to protective immunity
Authors: Migliorini Paola , Betschart Bruno , Corradin Giampietro
Published: March 2007

The immunogenicity of the non‐repetitive sequences of the Plasmodium berghei circumsporozoite (CS) protein was studied using synthetic peptides. Two CS sequences (residues 20‐39 and 57‐70) exhibiting T cell helper activity were identified. Immunization of BALB/c mice with a branched peptide containing either the 20‐39 or the 57‐70 sequence and two repeats (B epitope) in a linear sequence induced high titers of anti‐repeat and anti‐sporozoite antibodies. Mice immunized with the T‐B construct (high antibody titers) or with the 57‐70 epitope alone (no serum anti‐repeat or anti‐peptide antibodies) were protected to a similar degree after challenge with infective sporozoites. No protection was obtained in mice immunized with the 20‐39 epitope. These results indicate that BALB/c mice can be protected either by effector T cells or by high levels of anti‐repeat antibodies. Thus, in the same strain, a double mechanism of protection can be obtained by a synthetic peptide vaccine.
Prevention of experimental autoimmune arthritis with a peptide fragment of type II collagen
Authors: Ku Grace , Kronenberg Mitchell , Peacock Derek J. , Tempst Paul , Banquerigo Mona Lisa , Braun Benjamin S. , Reeve Joseph R. , Brahn Ernest
Published: March 2007

Collagen arthritis is induced in inbred rats with the injection of native type II collagen. The pathogenesis of this experimental autoimmune disease is T cell dependent. This study demonstrates that collagen‐specific Tcells, derived from pathogenic and nonpathogenic rat Tcell lines, both recognize the same peptide epitope. The epitope, consisting of amino acids 58–73 of cyanogen bromide fragment 11 of type II collagen, was as effective as whole collagen in stimulating a panel of collagen‐specific rat/mouse Tcell hybridomas. This peptide may, therefore, constitute a dominant epitope for CD4+ rat Tcells in their response to type II collagen. Administration of the peptide to either neonatal or adult rats prevented the subsequent induction of experimental arthritis with whole collagen, demonstrating that the in vivo response to this dominant epitope is, therefore, relevant in the pathogenesis of arthritis. Despite its ability to prevent collagen‐induced arthritis, administration of this peptide in incomplete Freund's adjuvant intradermally did not induce disease.
Diversity of immunoglobulin χ light chain gene rearrangements and evidence for somatic mutation in VχIV family gene segments in X‐linked agammaglobulinemia
Authors: Timmers Erik , Hermans Marcel M. , Kraakman Margriet E. M. , Hendriks Rudolf W. , Schuurman Ruud K. B.
Published: March 2007

X‐linked agammaglobulinemia (XLA) is a humoral immunodeficiency disease in man, characterized by an arrest in B lymphocyte differentiation at the precursor B cell stage. The structure of expressed immunoglobulin (Ig) χ light (L) chain rearrangements of nine B lymphoblastoid cell lines from one XLA patient was investigated by amplification of cDNA by the polymerase chain reaction using 5′ Vχ family‐specific primers and a 3′ χ constant region primer. Members of all four Vχ gene families were found to be utilized in Ig χ L chain rearrangements at frequencies that were consistent with random Vχ family usage. There was no preference for usage of any particular χ joining segment. Additional diversity was generated by deletions and random nucleotide insertions at the site of juxtaposition. Particular Vχ members seemed to be overrepresented in the sample. The observed homology of the VχI, VχII and VχIII region sequences, both to each other and to known germ‐line Vχ sequence indicated the absence of somatic mutations in the majority of these expressed Ig genes. In contrast of the single‐member VχIV family four different sequences were found to be expressed. That these sequences were mutated derivatives of a germ‐line VχIV element was substantiated both by sequence analysis and oligonucleotide hybridization. This finding shows that the mutation process can occur in early stages of B cell development i.e. before H chain class switch has occurred. The presence of these mutations is probably independent of clonal expansion since XLA patients are unable to respond to antigen. We conclude that the differentiation arrest in XLA does not preclude early onset of somatic mutation events in Vχ gene segments.
Skewing to the LFA‐3 adhesion pathway by influenza infection of antigen‐presenting cells
Authors: Van Kemenade Folkert J. , Kuijpers Karel C. , de Waal‐Malefijt René , Van Lier René A. W. , Miedema Frank
Published: March 2007

The effect of influenza (FLU) infection on heterotypic conjugate formation between antigen‐presenting cells and T lymphocytes has been studied with FLU‐specific T cell clones and FLU‐infected B‐lymphoblastoid cells (B‐LCL). Conjugate formation between FLU‐infected B‐LCL (FLU+ B‐LCL) and T cells was found to be consistently enhanced in comparison with peptide‐sensitized or uninfected B‐LCL. Treatment of B‐LCL with exogenous neuraminidase (NA‐NAse) similarly enhanced conjugate formation indicating that increased conjugate formation may be mediated by the viral neuraminidase. Monoclonal antibody blocking experiments revealed that the contribution by CD2/LFA‐3 is increased relative to that of LFA‐l/ICAM‐1 in conjugates between FLU+ B‐LCL or NANAse‐treated B‐LCL and T cell clones. In contrast, both pathways of adhesion contributed equally to conjugate formation between peptide‐sensitized B‐LCL or control B‐LCL and T cell clones. Thus, FLU infection causes increased conjugate formation between antigen‐presenting cells and T cells and skews towards CD2/LFA‐3‐dependent adhesion, independent of T cell receptor signalling.
Chimeric Fc receptors identify immunoglobulin‐binding regions in human FcγRII and FcϵRI
Authors: Hulett Mark D. , McKenzie Ian F. C. , Hogarth P. Mark
Published: March 2007

FcγRII and FcϵRI are functionally distinct cell surface receptors for immunoglobulin (Ig); FcγRII binds IgG with low affinity, whereas FcϵRI binds IgE with high affinity, yet they are homologous in structure and sequence having extracellular regions containing two Ig‐like domains with 38% amino acid identity. Chimeric receptors derived from human FcγRII and FcγRI were produced by exchanging homologous regions of the two receptors to define binding region(s) for IgG in FcγRII and IgE in FcϵRI. Firstly, a chimeric form of the FcϵRI α chain was produced by replacing the transmembrane region and cytoplasmic tail with that of FcγRII. This mutant α chain could be expressed on the cell surface independently of associated β and γ subunits, and retained high‐affinity IgE binding, indicating that the extracellular region of the FcγRI α chain is sufficient for high‐affinity IgE binding. Secondly, to identify the role of the individual domains in Fc binding of both FcγRII and FcγRI, chimeric receptors were generated by exchanging the first extracellular domains between FcγRII and the α chain mutant and used to demonstrate that the second extracellular domain of both receptors contains region(s) directly involved in Ig binding. Additional chimeric receptors were constructed to localize the Ig interactive regions in domain two of FcγRII and FcγRI; these identified a single region of IgG binding in FcγRII located between residues Ser136 to Val169, and at least three independent IgE binding regions in the FcγRI α chain, between residues Trp87 to Lys128, Tyr129 to Asp145, and Ser146 to Val169.
Expression and function of HLA‐B27 in lipid‐linked form: Implications for cytotoxic T lymphocyte‐induced apoptosis signal transduction
Authors: Gao Xiao‐Ming , Quinn Cheryl L. , Bell John I. , McMichael Andrew J.
Published: March 2007

Major histocompatibility complex (MHC) class I antigen‐restricted cytotoxic T lymphocytes (CTL) kill their target cells not only by inducing irreversible membrane damage but also by triggering a programmed suicide cascade (apoptosis) in target cells. Recent evidence suggests that MHC class I antigens are involved in apoptosis signal transduction in T cells. Therefore, it is possible that MHC class I antigens are also responsible for CTL‐induced signal transduction in target cells leading to apoptosis. To test this hypothesis, we have expressed HLA‐B27 in Chinese hamster ovary (CHO) cells in a phosphatidyl inositol (PI) anchored form. The expressed Pl‐anchored HLA‐B27 (PI‐B27), a 42‐kDa molecule which can be cleaved off the cell surface by Pi‐specific phospholipase C, can function as an MHC restriction and antigen presentation element for specific CTL. Furthermore, PI‐B27 transfectant CHO cells undergo rapid DNA fragmentation when pulsed with the appropriate peptide and treated with specific CTL, suggesting that the cytoplasmic and transmembrane domains of the heavy chain of class I MHC molecules are not required in CTL‐induced apoptosis signal transduction in target cells.
Antisense oligonucleotides to interleukin‐4 regulate IgE and IgG2a production by spleen cells from Nippostrongylus brasiliensis‐infected rats
Authors: Benbernou Naïma , Matsiota‐Bernard Peggy , Guenounou Moncef
Published: March 2007

Interleukin‐4 (IL‐4) plays a key role in the regulation of immunoglobulin production. In the present study, we examined the role of IL‐4 in the production of IgE and IgG2a by spleen cells from Nippostrongylus brasiliensis‐infected rats using the antisense oligonucleotide strategy. Three antisense oligonucleotides (15 and 20 nucleotides) were selected near the ATG initiation codon of the murine IL‐4 gene. Significant and reproducible inhibitions of IgE and IgG2a production were observed with the addition of two different oligonucleotides, the most inhibitory being specific for 15 nucleotides corresponding to the downstream AUG sequence oligodeoxynucleotide (ODN‐1), while the corresponding sense sequence had no effect. The inhibition was directly related to the ODN‐1 concentration on IgE response, and IgG2a production was more sensitive to the inhibitory effect of ODN‐1 than the IgE response. Our results suggest that the ODN‐1 effect is rather specific for the IL‐4 gene since the production of other cytokines was not modified and the exogenous addition of IL‐4 reversed the ODN‐1 effect. In contrast to IgE and IgG2a, ODN‐1 induced a significant enhancement of IgM synthesis. The use of a phosphorothioate ODN of the same sequence as ODN‐1 resulted in a greater inhibitory effect on IgE and IgG2a production. These data indicate that IL‐4 contributes to the spontaneous production of IgE and IgG2a, and argue that antisense strategy constitutes a useful tool for the regulation of antibody response.
Activation of mucosal Vβ3+ T cells and tissue damage in human small intestine by the bacterial superantigen, Staphylococcus aureus enterotoxin B
Authors: Lionetti Paolo , Spencer Jo , Breese Emma J. , Murch Simon H. , Taylor Jacqueline , Macdonald Thomas T.
Published: March 2007

Staphylococcus aureus enterotoxin B (SEB) was added to explants of fetal human intestine in organ culture or administered into the lumen of fetal small intestine prior to culture. Both routes produced a massive increase in lamina propria T cells expressing Vβ33, and to a lesser extent, those expressing Vβ5 and Vβ12. SEB‐activated lamina propria T cells produced interleukin‐2 and interferon‐Y and T cell activation was accompanied by tissue damage, which was inhibited by FK506.
Tolerance induction by clonal deletion of CD4+8+ thymocytes in vitro does not require dedicated antigen‐presenting cells
Authors: Pircher Hanspeter , Brduscha Karin , Steinhoff Ulrich , Kasai Michiyuki , Mizuochi Toshiaki , Zinkernagel Rolf M. , Hengartner Hans , Kyewski Bruno , Müller Klaus‐Peter
Published: March 2007

The cellular requirements of T cell tolerance induction in the thymus by clonal deletion was investigated by using an in vitro assay: thymocytes from mice expressing a transgenic TcR specific for lymphocytic choriomeningitis virus (LCMV) and H‐2Db were co‐cultured with various H‐2b cell types as antigen‐presenting cells in the presence of the antigenic LCMV peptide. The results revealed that all cell lines examined including embryonic and transformed fibroblasts, melanoma cells, cortical thymic epithelial cells, lymphomas and neuronal cells induced an antigen dose‐dependent deletion of CD4+8+ thymocytes. Similarly, highly enriched accessory cell populations from thymus and spleen (macrophages, dendritic and cortical epithelial cells, i.e. thymic nurse cells) could induce antigen‐specific depletion of immature CD4+8+ thymocytes. Depending on the cell type examined micromolar to picomolar concentration of LCMV peptide were required to induce deletion. The effectiveness of deletion by the different cell types did not correlate with their major histocompatibility class I expression level; it was, however, influenced by the presence of ICAM‐1 adhesion molecules.
Dissociation of natural killer and lymphocyte‐activated killer cell lytic activities in human CD3− large granular lymphocytes
Authors: Clément Marie‐Véronique , Legros‐Maida Sabine , Soulie Annie , Jacqueline Guillet  , Sasportes Marilyne
Published: March 2007

CD3− large granular lymphocytes (LGL) are known to display natural killer cell (NK) activity without prior sensitization or restriction by major histocompatibility antigens. Upon short‐term exposure to interleukin‐2, NK cells were shown to acquire lymphocyte‐activated killer cell (LAK) activity. The aim of this study was to analyze the characteristics of these lytic activities. Our data indicated that both NK and LAK activities were Ca2+ dependent; however, they could be dissociated by a Ca2+ channel blocker or a Ca2+ channel competitor agent. Moreover, NK activity was associated with granule exocytosis of lytic proteins spontaneously present in CD3− LGL, the most likely candidate being the pore‐forming protein perforin. By contrast, LAK activity was found to be dependent on de novo protein synthesis and distinct from granule exocytosis. Our results strongly suggest that NK and LAK activities could be defined as two distinct pathways involving different lytic mediators.
Treatment with anti‐CR3 antibodies ED7 and ED8 suppresses experimental allergic encephalomyelitis in Lewis rats
Authors: Huitinga Inge , Damoiseaux Jan G. M. C. , Döpp Ed A. , Dijkstra Christine D.
Published: March 2007

Experimental allergic encephalomyelitis (E AE) is an inflammatory disease of the central nervous system (CNS). Among the leukocytes which infiltrate the CNS during EAE, numerous macrophages are present. These macrophages are thought to play a crucial role in the generation of tissue damage and attendant neurological deficits. The mechanism by which the macrophages migrate across the blood‐brain barrier is not yet clear. Membrane proteins involved in macrophage adherence to the endothelium include the CD11b/CD18 integrin, also known as the type 3 complement receptor (CR3). In this study we show that two monoclonal antibodies (mAb) ED7 and ED8 are directed against rat CR3. In addition, these mAb reduce recruitment of myelomonocytic cells towards thioglycollate induced peritonitis by 15‐33%. This indicates that both ED7 and ED8 interfere with an epitope on CR3, which is involved in recruitment of phagocytes towards inflammatory lesions. Intravenous injection of ED7 and ED8 suppressed clinical signs of EAE. MRC OX‐42, which also recognizes CR3, did not reduce thioglycollate‐induced phagocyte recruitment into the peritoneum, and had no effect on EAE. These findings suggest that CR3 plays a role in the recruitment of macrophages towards the inflamed CNS of EAE animals, and confirm the role of macrophages in the generation of clinical signs of EAE. Involvement of CR3 in other phagocyte immune functions during EAE is discussed.
The murine T cell line CT6 provides a novel bioassay for interleukin‐7
Authors: Willcocks Joanie L. , Hales Anne , Page Theresa H. , Foxwell Brian M. J.
Published: March 2007

Like interleukin (IL)−2 and IL‐4, IL‐7 can act as a growth factor for activated T lymphocytes. Upon screening a panel of growth factor‐dependent T cell lines, we found that only the cell line CT6 responded to IL‐7, indeed as vigorously as to IL‐2. Obviously, these findings challenge the validity of previous results on IL‐2 production obtained using the CT6 cell line. However, they also demonstrate a novel and sensitive system for the bioassay of IL‐7. The ability of the surveyed T cell lines to proliferate to IL‐7 corresponded with the expression of IL‐7 receptors (IL‐7R) on the cell surface. The murine IL‐7R on CT6 was shown to bind IL‐7 with dual affinity and was visualized as an affinity cross‐linked complex of 93 kDa. This IL‐7R appears similar to that seen on murine splenic T cells and on 70Z/3, the pre‐B cell line from which the murine IL‐7R was cloned.
Chicken B cells adhere to the CS‐1 site of fibronectin throughout their bursal and postbursal development
Authors: Palojoki Eeva , Toivanen Paavo , Jalkanen Sirpa
Published: March 2007

In chickens, the pre‐B cell development takes place in a specialized organ, the bursa of Fabricius. In this study, we show that fibronectin can be detected in the bursa at least from the day 15 of embryonic incubation up to 10 weeks of age after which the bursa starts to involute. During the embryonic incubation, fibronectin localizes mainly in the areas between the follicles and later on also in the border zone between the cortical and medullary parts of the lymphoid follicles. By adhesion studies with synthetic peptides we show that immature bursal B cells bind in a dose‐dependent manner to the CS‐1 site of the type III connecting segment at the carboxy‐terminal end of fibronectin throughout their bursal development. Postbursal B cells from chicken spleen also display a similar preference of binding to the CS‐1 site of fibronectin, whereas peripheral blood B lymphocytes bind to the 40‐kDa fragment of fibronectin, but do not adhere as strongly to the CS‐1 site. Instead, they recognize to some extent also the heparin‐binding YEKPGSPPREVVPRPRPGV peptide. The adhesion of the bursal B cells to the CS‐1 site can partially be blocked by preincubation of cells with a synthetic CS‐1 peptide and also by preincubation with a monoclonal anti‐fibronectin receptor antibody. In contrast, the RGD motif present in several adhesion‐associated stromal molecules did not promote any adherence of bursal lymphocytes. The bursal B cells which adhere to the fibronectin molecule in vitro form only a small fraction of the total number of B cells present in the bursa as only less than 4% of the cells remained bound to the surface coated with fibronectin in the adhesion studies. In conclusion, these results suggest that fibronectin may be involved in the differentiation of pre‐B cells in the bursa of Fabricius and also in the adherence of postbursal B cells in the spleen.
Soluble T cell receptor‐like properties of an HLA‐B35‐specific monoclonal antibody (TÜ165)
Authors: Uchańska‐Ziegler Barbara , Nößner Elfriede , Schenk Andrea , Ziegler Andreas , Schendel Dolores J.
Published: March 2007

A mouse monoclonal antibody of IgM class (TÜ165) was produced using Epstein‐Barr virus (EBV)‐infected mutant cells derived from the human BJAB‐B95.8.6 cell line as immunogen. Binding studies with several HLA deletion mutant cell lines indicated that TÜ165 recognized the HLA‐B35 molecule. In a panel of 89 EBV‐transformed lymphoblastoid cell lines, all HLA‐B35+ cells (n = 24) reacted with TÜ165 while all but two HLA‐B35− lines (n = 65) were unreactive (r = 0.95). Surprisingly, peripheral blood lymphocytes of HLA‐B35+ donors were unreactive; however, strong enhancement of TÜ165 recognition was observed with B cells of one of these individuals after transformation with EBV (B95.8 strain). Transfection of both HLA‐B35 and human ß‐microglobulin genomic DNA into mouse P815 cells led to high expression of HLA‐B molecules; yet, expression of the TÜ165 epitope was not observed. Furthermore, the EBV‐negative cell line BJAB as well as the EBV‐infected (P3HR1 strain) line BJAB‐HR1K were only weakly reactive, whereas the BJAB‐B95.8 cell line was strongly positive. These results indicate that EBV‐encoded or ‐controlled peptide(s) must be bound by HLA‐B35 antigens to create the epitope which allows efficient binding of TÜ165.
CD 69 expression during selection and maturation of CD4+8+ thymocytes
Authors: Swat Wojciech , Dessing Mark , Boehmer Harald Von , Kisielow Pawel
Published: March 2007

We have analyzed the inducibility of protein kinase C (PKC)‐dependent expression of CD 69 molecules in T cell receptor (TCR) transgenic thymocytes developing in the presence or absence of selecting, class I major histocompatibility complex (MHC) molecules. Small CD4+8+ thymocytes developing in the absence of selecting MHC molecules could not be induced to express CD 69 by TCR cross‐linking even after spontaneous in vitro up‐regulation of their TCR level which resulted in enhanced Ca++ flux. In contrast, a small proportion of CD4+8+TCRlow and most TCRhigh (CD4+8+ and CD4+8+) thymocytes developing in the presence of selecting MHC ligands could be induced to express CD 69 upon TCR cross‐linking. Unlike the anti‐TCR antibody, phorbol 12‐myristate 13‐acetate ‐ a direct activator of PKC ‐ induced the expression of CD 69 on all thymocytes. These results suggest that positive selection of CD4+8+ thymocytes results in coupling of TCR‐mediated signals to the CD 69 expression pathway. In vitro analysis of thymocytes before and after positive selection suggests that (1) positive selection does not immediately result in resistance to deletion and (2) that sustained TCR ligation is needed to promote maturation of positively selected CD4+8+ thymocytes resulting in gradual loss of the sensitivity to deletion and acquisition of the ability to proliferate in response to TCR‐mediated signals.