bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of a protective immunodominant B cell epitope of pertactin (P.69) from Bordetella pertussis
Authors: Charles Ian G. , Li Jingli , Roberts Mark , Beesley Katrina , Romanos Mike , Pickard Derek J. , Francis Mike , Campbell Dick , Dougan Gordon , Brennan Michael J. , Manclark Charles R. , Jensen Maria Au , Heron Ivor , Chubb Ann , Novotny Pavel , Fairweather Neil F.
Published: March 2007

Epitopes defined by monoclonal antibodies (mAb) specific for the Bordetella pertussis outer membrane protein P.69 (pertactin) were mapped using a series of amino‐ and carboxy‐terminal deletion mutants expressed in Escherichia coli. mAb were found to bind predominantly to a region of pertactin spanning a (Pro‐Gln‐Pro)5 repeat motif and one mAb was found to bind to another region spanning a (Gly‐Gly‐Xaa‐Xaa‐Pro)5 repeat motif. To localize further the mAb‐binding sites, a panel of synthetic peptides, a series of 94 overlapping hexameric peptides, and a P.69 30‐amino acid fusion to a hepatitis B core protein (HBcAg‐69), were synthesized. This combined approach has identified the binding site for the mAb BBO5: Pro‐Gly‐Pro‐Gln‐Pro‐Pro; mAb BBO7, E4A8 and E4D7: Ala‐Pro‐Gln‐Pro‐Pro‐Ala‐Gly‐Arg; and mAb BPE3: Thr‐Leu‐Trp‐Tyr‐Ala‐Glu‐Ser‐Asn‐Ala‐Leu‐Ser‐Lys‐Arg. We have used a non‐lethal murine respiratory model of B. pertussis infection to investigate the ability of a peptide containing the epitope of the mAb BBO5 to elicit protective immunity. Immunization of mice with the HBcAg‐69 protein prevented growth of B. pertussis in the lungs compared to mice receiving HBcAg alone, and protection correlated with high titers of anti‐P.69 antibodies.
Biochemical properties of MHC class II molecules endogenously synthesized and expressed by mouse Langerhans cells
Authors: Becker Detlef , Reske‐Kunz Angelika B. , Knop Jürgen , Reske Konrad
Published: March 2007

The cell surface expression and biosynthesis of Langerhans cells (LC)‐derived major histocompatibility complex (MHC) class II molecules from epidermal cells (EC) prepared freshly and cultured for up to 3 days was investigated. Based on the constitutive expression of MHC class II determinants by LC, a panning and magnetic bead selection procedure was employed, yielding 65% and 86% of I‐A+ cells, respectively. Phenotypical and cytochemical examinations revealed that the two LC preparations were free of contaminating macrophages as well as B and T cells. Freshly prepared enriched LC were highly efficient in the stimulation of protein antigen‐specific T cell clones, while LC purified from short‐term cultured EC suspensions proved to be more efficient allogeneic stimulator cells than fresh LC. Comparative analysis of LC obtained from freshly prepared and from short‐term‐cultured EC preparations indicated an up‐regulation of MHC class II determinants during short‐term culture. Radioiodination analysis of LC selected by magnetic beads demonstrated prominent class II α and β chain signals with only a minute fraction of invariant chains p35 and p45 being expressed at the cell surface. Unlike class II complexes derived from B cells, those from LC contained invariant chain fragment p20 in association with α/β heterodimers at the plasma membrane. No qualitative differences between freshly isolated and 3‐day cultured LC in cell surface expressed MHC class II components were detectable. Metabolic labeling with subsequent two‐dimensional electrophoresis revealed distinct features of LC‐derived MHC class II molecules with a high proportion of invariant chains in particular γ and p40 and their extensive sialylation. While fresh and 1‐day cultured LC exhibited appreciable levels of newly synthesized class II molecules, a dramatic down‐regulation in class II and invariant chain synthesis was measured after 3 days of continuous in vitro culture.
Human major histocompatibility complex class II‐negative colon carcinoma cells present staphylococcal superantigens to cytotoxic T lymphocytes: evidence for a novel enterotoxin receptor
Authors: Dohlsten Mikael , Hedlund Gunnar , Segren Sverker , Lando Peter A. , Herrmann Thomas , Kelly Adrian P. , Kalland Terje
Published: March 2007

The staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules on target cells and activate T cells expressing particular T cell receptor Vβ sequences. In this report we demonstrate that SE bind to the MHC class II− SW620, Colo320DM and WiDr human colon carcinoma cell lines and direct cytotoxic T lymphocytes (CTL) to mediate strong target cell killing. Flow cytometry analysis, immunoprecipitation and Northern blotting experiments failed to demonstrate any surface expression of HLA‐DR, HLA‐DP and HLA‐DQ isotypes on the SW620 colon carcinoma cell line, whereas abundant expression of these isotypes was seen on Raji cells. SEB and SEC1 were efficiently presented at picomolar concentration by the MHC class II− colon carcinoma cells and MHC class II+ Raji cells, whereas SEA and SED were preferentially presented on the MHC class II+ Raji cells. An anti‐HLA‐DR monoclonal antibody inhibited SEB‐induced CTL targeting to Raji, but did not influence the killing of SW620 cells. Our data suggests the existence of functionally active SE‐binding structures on human colon carcinoma cells which are distinct from the conventional MHC class II molecules. The possibility that these putative new SE receptors play a role in the enterotoxin action of SE must be considered.
Sequences of the mouse F protein alleles and identification of a T cell epitope
Authors: Schofield J. Paul , Vijayakumar R. K. , Oliveira David B. G.
Published: March 2007

F protein is found predominantly in the liver and is of unknown function. The protein has been of interest to immunologists in the areas of self tolerance and the immunogenetics of the anti‐F protein response. In the mouse there are two alleles (F1 and F2), and although mice are completely tolerant to the self form of the protein, mice of responder strains make a good antibody response to immunization with the non‐self form. This response cross‐reacts with the self form, implying firstly, that autoreactive B cells are present and that tolerance is therefore maintained at the T cell level, and secondly, that the difference between the two allelic products defines a T cell epitope.

Primers based on the published sequence for rat F protein were used in the polymerase chain reaction to amplify the cDNA for the two mouse alleles. Subsequent sequencing shows a high degree of sequence identity between the rat and mouse cDNA. The two mouse cDNA are identical apart from a single A to G base change which predicts an asparagine (F1 protein) to aspartate (F2 protein) amino acid residue change. Using allele‐specific oligonucleotide probes we confirmed that this base change has the same strain distribution as the previously determined F protein type. Isoelectric focusing shows that F1 protein migrates in a more basic position than F2 protein, as predicted by the asparagine to aspartate change. Finally, a synthetic peptide from the allovariable site of F2 protein will successfully restimulate T cells in vitro from an F1 type mouse primed in vivo with whole F2 protein, whereas the corresponding peptide from F1 protein will not. This is evidence that, as predicted, the allovariable site does indeed define a T cell epitope. Peptides covering the rest of the F2 protein molecule were not stimulatory.
Interleukins 1, 4 and 6 induce the colony formation of human bone marrow B lineage cells
Authors: McGinnes Kimberley , Paige Christopher J.
Published: March 2007

An agar‐based, B cell colony assay (McGinnes, K. et al., Blood 1990, 76: 896) has been used to study the influence of known cytokines on the growth of B lineage colonies initiated by cells from normal, human bone marrow samples. We demonstrate that a combination of interleukin (IL) 1, IL 4 and IL 6 act directly to promote the generation of plaque‐forming colonies. IL 6 was shown to act at a late stage of colony formation, which is consistent with its role in the induction of immunoglobulin secretion from mature B cells. In contrast, IL 1 and IL 4 were required at earlier stages in the formation of colonies containing cells which secrete immunoglobulin.
Cell surface expression of the short immunoglobulin μ chain (Dμ protein) in murine pre‐B cells is differently regulated from that of the intact μ chain
Authors: Tsubata Takeshi , Tsubata Rieko , Reth Michael
Published: March 2007

Pre‐B cells carrying DJH rearrangements in an appropriate reading frame produce a short μ protein consisting of a DJH and the Cü & sequence (ü protein). We analyzed a Dμ‐producing Abelson‐murine leukemia virus‐transformed murine pre‐B line, 300–19, and demonstrated that Dμ proteins are expressed on the cell surface in association with surrogate L chain proteins (Λ5 and VpreB). However, when we introduced an expression vector coding for the Dμ protein into the null pre‐B line P17–27, which produce Λ5 and VpreB but no Ig molecules, most of the cells did not express Dμ proteins on the cell surface although Dμ proteins were produced intracellularly. On the other hand, P17–27 brings intact μ chains on the cell surface, when a vector coding for the intact μ chain is introduced. Thus, cell surface expression of the Dμ protein has different requirements from that of the intact μ chain. A possible role of the VH protein encoded by germ‐line VH transcripts is discussed.
Interleukin 1‐induced maturation of progenitor thymocytes
Authors: Takashi Tohru , Gause William C. , Wilkinson Miles , Macleod Carol L. , Steinberg Alfred D.
Published: March 2007

Regulation of thymocyte development was assesed by culturing purified CD4−CD8− thymocytes with cytokines. Sorted CD3−CD4−CD8− adult thymocytes responded to the combination of interleukin (IL) 1 plus IL 2 without additional mitogens or co‐mitogens with both cellular proliferation and cell surface expression of the T cell receptor (TcR)/CD3 complex. IL 2 alone induced neither proliferation nor cell surface TcR/CD3 expression. IL 1 alone was sufficient to induce cell surface TcR/CD3 without proliferation. Prior to stimulation with cytokines, the progenitor CD4−CD8− thymocytes accumulated TcR β and CD3 γ, δ, ε and ϱ, mRNA but TcR α mRNA was not detectable. Stimulation with IL 1 led to a dramatic induction of TcR α mRNA without an increase in the other transcripts. These studies suggest that IL 1 regulates the differentiation status of immature adult thymocytes. Nuclear run‐on studies suggested that the increase in TcR α mRNA accumulation induced by cytokines might result from post‐transcriptional accumulation.
Stimulation of B lymphocytes via CD72 (human Lyb‐2)
Authors: Kamal Mohammed , Katira Anita , Gordon John
Published: March 2007

A monoclonal antibody to CD72, the 45‐kDa human homolog of marine Lyb‐2, was found to augment selective activation pathways in tonsillar B lymphocytes. By itself, CD72 antibody provided a weak direct stimulus to resting B cells as assessed by [3H]dThd incorporation; this was modestly enhanced on the addition of interleukin 4 (IL 4) or following ligation of surface CD40. CD72‐delivered signals were more evident in co‐stimulation assays with phorbol ester and with a synergistic combination of IL 4 and CD40 antibody, but not with calcium ionophore or a CD23 antibody; rapidly cycling B cells were refractory to signaling via CD72 whether or not other co‐stimuli were present. A unique feature of the CD72‐delivered signal was its ability to enhance synergistically stimulations triggered with immobilized antibody to IgM, while failing to augment responses initiated by soluble anti‐μ. On direct culture of resting B lymphocytes with CD72 antibody, an approximately twofold increase in the expression of major histocompatibility complex class II DQ antigen was observed, an augmentation similar to that achieved with IL 4; CD72 antibody also mimicked IL 4 in its ability to drive G0 cells into the early G1 phase of cell cycle. In contrast to IL 4‐promoted stimulation, CD72 antibody failed to up‐regulate the surface expression of either IgM or the CD23 antigen. CD72 expression itself was found to be weak on resting B lymphocytes and was modestly enhanced following culture with IL 4. The findings are discussed with reference to observations made on the triggering of murine B lymphocytes through Lyb‐2 and within the context of previously defined human B cell activation pathways.
Identity of human Lyb‐2 and CD72 and localization of the gene to chromosome 9
Authors: von Hoegen Ilka , Hsieh Chih‐Lin , Scharting Roland , Francke Uta , Parnes Jane R.
Published: March 2007

We have recently reported the isolation of cDNA clones encoding the human homolog of the mouse B cell differentiation antigen Lyb‐2. Expression of Lyb‐2 is restricted to B lineage cells and turned off in antibody‐secreting plasma cells in both mice and humans. Functional studies with anti‐mouse Lyb‐2 monoclonal antibodies (mAb) suggest that this protein may be involved in signals for B cell proliferation. We now describe the generation of mAb specific for human Lyb‐2. Furthermore, we demonstrate that the human Lyb‐2 protein is recognized by a mAb specific for the newly clustered pan B cell surface antigen CD72, which had been defined by mAb. Mouse L(tk−) cells expressing a transfected cDNA encoding human Lyb‐2 bind a mAb specific for CD72. We further show that an antiserum and a mAb specific for human Lyb‐2 and an anti‐CD72 mAb immunoprecipitate the identical protein from human splenic B cells and B cell lines, as well as from transfected L(tk−) cells. These data indicate that CD72 is the human equivalent of mouse Lyb‐2. We have additionally localized the gene for human Lyb‐2/CD72 on the short arm of chromosome 9. Mouse Lyb‐2 had been previously mapped to mouse chromosome 4. Lyb‐2/CD72 is now the tenth gene known to be localized on human chromosome 9 and mouse chromosome 4.
Autoreactive T and B cells responding to myelin proteolipid protein in multiple sclerosis and controls
Authors: Sun Jia‐Bin , Olsson Tomas , Wang Wei‐Zhi , Xiao Bao‐Guo , Kostulas Vasilios , Fredrikson Sten , Ekre Hans‐Peter , Link Hans
Published: March 2007

The pathogenesis of multiple sclerosis (MS) could involve an autoimmune response to proteolipid protein (PLP). Immunization of experimental animals with this major myelin protein can lead to experimental allergic encephalomyelitis. To identify a possible role of PLP as target antigen in MS, we evaluated T celi immunity to PLP in blood and cerebrospinal fluid (CSF) from patients with MS and controls by counting cells which in response to PLP in short‐term cultures secreted interferon‐γ. The PLP‐specific B cell response was analyzed by counting cells secreting anti‐PLP antibodies. PLP‐reactive T cells were detected in blood of most MS patients (mean value 1 per 20408 mononuclear cells), and at 41‐fold higher numbers in CSF (mean 1 per 500 CSF cells). Anti‐PLP IgG antibody‐secreting cells were detected in blood from most MS patients (mean 1 per 30303 cells), but such cells were 49‐fold more frequent in CSF (mean 1 per 625 cells). PLP‐reactive T and B cells were also detected in blood and CSF from control patients, but at much lower numbers. A strong and persistent autoimmune response to PLP as well as to other myelin proteins, enriched in CSF, is proposed to be pathogenetically important in MS.
Molecular characterization of an IgA receptor from group B streptococci: sequence of the gene, identification of a proline‐rich region with unique structure and isolation of N‐terminal fragments with IgA‐binding capacity
Authors: Hedén Lars‐Olof , Frithz Elisabet , Lindahl Gunnar
Published: March 2007

Certain strains of group B streptococci express a cell surface protein that binds IgA and acts as a virulence factor. This IgA receptor is referred to here as protein Bac. The gene for protein Bac was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. The deduced amino acid sequence of 1134 residues includes a signal sequence of 37 amino acids and a putative membrane anchor region at the C‐terminal end. The processed form of the receptor, 1097 residues, has a calculated molecular weight of 123 786. There are no cysteines in protein Bac, suggesting a fibrillar structure. The C‐terminal half of the protein includes a 90 residues long region with a novel type of periodic structure, the “XPZ motif”, in which every third amino acid is proline. Unlike other bacterial immunoglobulin‐binding proteins, there are no long repeats in protein Bac. Clones which express only part of the protein Bac gene were used to show that IgA‐binding takes place in the N‐terminal part of the molecule. Protein Bac was originally described as an antigen called β, but N‐terminal fragments that bind IgA do not react with a reference serum against the β antigen. These and other data indicate that protein Bac can be divided into two regions with different functions: an N‐terminal IgA‐binding region and a C‐terminal region corresponding to the β antigen. The IgA‐binding region of protein Bac does not show any homology to protein Arp, the IgA receptor from group A streptococci, although these receptors have similar binding properties. This indicates that convergent evolution has favored the appearance of these two structurally different streptococcal IgA receptors.
Molecular heterogeneity of the 1.0‐kb Tβ transcript in natural killer and γ/δ lymphocytes
Authors: Fagioli Marta , Care Alessandra , Ciccone Ermanno , Moretta Lorenzo , Moretta Alessandro , Meccia Ettore , Testa Ugo , Fallnl Brunangelo , Grignani Fausto , Peschle Cesare , Pelicci Pier Giuseppe
Published: March 2007

The T cell receptor (TcR) β chain is encoded by a 1.3‐kb mRNA which contains variable (V), diversity (D), joining (J) and constant (C) regions, A shorter 1.0‐kb transcript with C but no V sequences is found in thymocytes, α/β and γ/δ T lymphocytes and natural killer cells. The origin, clonal variability and function of this transcript is unknown. We isolated cDNA clones representative of the 1.0‐kb mRNA from cDNA libraries of either polyclonal or clonal natural killer and γ/δ lymphocytes. Ten different types of cDNA clones belonging to three separate groups were identified: (a) “J‐C clones” consisting of one of five Jβ regions and the corresponding Cβ1 or Cβ2 regions; (b) “D‐J‐C clones” composed of Dβ1/or Dβ2/Jβ rearranged sequences and Cβ1 or Cβ2 sequences; (c) “5′C clones” made up of Cβ1 or Cβ2 regions preceded by the corresponding genomic 5′ flanking region. The presence of recombination signal sequences in J‐C and D‐J‐C clones suggests that the first derive from mRNA transcribed from promoters located in the 5′ J region and the second from mRNA transcribed from promoters situated in the 5′ D region. Nucleotide sequence analysis demonstrated that only three of the ten types of clones isolated had the potential to code a short cytoplasmic Tβ protein with a variable D‐Jβ N terminus. These findings have implications for the mechanisms of Tβ germ‐line transcription and the function of the Tβ transcripts.
The nature and importance of the inter‐ε chain disulfide bonds in human IgE
Authors: Helm Birgit A. , Ling Yan , Teale Catherine , Padlan Eduardo A. , Brüggemann Marianne
Published: March 2007

IgE antibodies are best known for their pathological role in allergy. The class‐specific effector sites are located in the ε chains; these form covalent dimers via two cystine residues (Cys241 and Cys328) linking opposite Cε2 domains. The nature and biological significance of the inter‐ε chain disulfide‐bond arrangement is unresolved. For structural and functional analysis site‐specific mutations were introduced into the Cε2 domain of recombinant human IgE. The introduction of an additional cyanogen bromide cleavage site (His246 → Met) facilitated the identification of parallel disulfide bond pairing. This linkage was also confirmed for myeloma IgE PS by sequence determination of disulfide‐linked C2 dimers. Substitution of Cys241 and Cys328 by Ser does not destroy receptor binding, but reductive alkylation, or the replacement of Cys328 by Met, leads to loss of activity. This shows that covalent dimerization is not essential for IgE/receptor interaction and points to the importance of the structural integrity of the site surrounding Cys328, visualized in a new model of human Fcε.
Induction of antibodies against a peptide hapten does not require covalent linkage between the hapten and a class II presentable T helper peptide
Authors: Sarobe Pablo , Lasarte Juan‐José , Golvano José , Gullón Arturo , Civeira Maria‐Pilar , Prieto Jesús , Borrás‐Cuesta Francisco
Published: March 2007

Following immunization with a complex antigen, a B cell internalizes this antigen through an interaction between its surface immunoglobulins and an epitope of the antigen. Enzymatic processing of the antigen frees one or more short peptide determinants (TD) which bind to class II molecules of the B cell. If the complex TD‐MHC II is recognized by the receptor of a T helper cell, T cell help is provided leading to the expansion of an antibody‐producing B cell clone specific for the epitope. We present experimental evidence proving that the induction of anti‐peptide hapten antibodies does not require covalent linkage between the peptide hapten and the peptide behaving as TD. Indeed, high anti‐peptide hapten antibody titers are induced if an emulsion of TD and hapten are injected in the same immunization site. This result suggests a way to manipulate antibody production with useful applications to research and therapy.
2,4,6‐Trinitrophenyl (TNP) responsiveness of anti‐TNP (Sp6) transgenic mice
Authors: Zöller Morgot
Published: March 2007

Transgenic mouse models have demonstrated clonal deletion as well as clonal anergy of monospecific, high‐avidity autoreactive B cell. The function and fate of naturally activated B cells, many of them displaying degenerate specificity including autoreactivity, are still a matter of debate. The question was pursued in Sp6‐transgenic mice. Sp6, a monoclonal anti‐2,4,6‐trinitrophenyl (TNP) IgM has been shown to react with a variety of self antigens. Responsiveness of antibody‐secreting B cells was followed throughout postnatal development of Sp6‐transgenic mice and was related to the availability of antigen‐ and idiotype‐specific help.

Thymus as well as spleen cells of transgenic mice contained a significantly higher number of TNP‐specific B cell than non‐transgenic controls. In contrast to control mice, the number of TNP‐specific B cells remained unchanged or decreased in thymus and spleen of transgenic mice after antigenic stimulation with TNP in T‐dependent (TD) and T‐independent (TI) form.

Since the relative frequency of transgenic B cells was in particular diminished after repeated stimulation with TD antigen, it was examined whether limited responsiveness was linked to the available repertoire of helper T cells. Early after birth of transgenic individuals, thymic as well as splenic T cells which proliferated in response to TNP and Sp6 and provided help for B cells were found to be significantly augmented. Their number decreased rapidly during postnatal maturation and Th cells did not expand after antigenic stimulation. There was no indication that in the naive host transgenic B cells would suppress proliferation of TNP‐ and Sp6‐specific T cells, but they did so after antigenic stimulation. Furthermore, and in contrast to B cells of non‐transgenic mice, transgenic B cells were unable to present nominal antigen in a stimulatory way.

The decrease in the number of B cells after antigenic stimulation indicated that autoreactive transgenic B cells may be subject to (functional) deletion under selected circumstances. In addition, idiotype‐ and antigen‐specific help was impaired in Sp6‐transgenic mice and this clearly was due to interactions with B cells expressing the immunoglobulin transgene.
Cloning and structural analysis of MMCP‐1, MMCP‐4 and MMCP‐5, three mouse mast cell‐specific serine proteases
Authors: Huang Ranyang , Blom Thomas , Hellman Lars
Published: March 2007

Here we present the cloning of three novel mouse mast cell‐specific serine proteases, MMCP‐1, MMCP‐4 and MMCP‐5. A region of approximately 4 kb covering the five exons and 930 bp 5′ and 280 bp 3′ flanking sequences of the gene for MMCP‐1 was characterized by nucleotide sequence analysis. A comparison with the corresponding region of the rat mucosal mast cell‐specific protease RMCP‐II is presented. cDNA clones for the mast cell proteases MMCP‐4 (950 bp) and MMCP‐5 (1098 bp) were isolated from a cDNA library of a connective tissue mast cell‐like mouse mastocytoma cell line. All three proteases were found to belong to the family of chymotrypic serine proteases as deduced from the absence of the Asp 189 which is characteristic for all serine proteases having cleavage specificities similar to pancreatic trypsin. The active polypeptides, excluding possible post‐translational glycosylations, have an Mr of 25–26 kDa. Analysis of the amino acid composition reveals a positive net charge for all three proteases (MMCP‐1 +3, MMCP‐4 +18 and MMCP‐5 +12). Based on their high sequence identity (88%) and high positive net charges (+18 and +18, respectively) we assume that the MMCP‐4 is the mouse homolog to rat RMCP‐I.

Probes specific for each of these three highly homologous protease genes have been generated by subcloning of fragments of approximately 100 bp in length, originating from the 3′ ends of the mRNA into plasmid vectors. Northern blot analysis of mRNA from a number of murine cell lines shows gene expression of these proteases to be specific for the differentiation stage of the mast cell. The MMCP‐1 is expressed only at the mucosal mast cell stage and and 5 only in mast cells of the connective tissue mast cell stage. These serine proteases may serve as highly specific markers in the analysis of mast cell heterogeneity, differentiation and function.
Immunodeficiency with low expression of the T cell receptor/CD3 complex. Effect on T lymphocyte activation
Authors: Le Deist Françoise , Thoenes Gabriela , Corado José , Lisowska‐Grospierre Barbara , Fischer Alain
Published: March 2007

We report the consequences of low expression of the T cell receptor (TcR)/CD3 complex by T lymphocytes from a 4‐year‐old boy with a mild immunodeficiency. TcR/CD3 expression was found to be deficient on both resting and activated T cells, using both anti‐CD3 and anti‐TcR α/β monoclonal antibodies. As shown by immunofluorescence and immunoprecipitation studies, residual expression (corresponding to about 10% of normal) was detectable on resting and activated TcR α/β+ T cells. Other T cell membrane receptors were normally expressed. The functional consequences of this TcR/CD3 expression deficiency included an absence of T cell proliferation, interleukin 2 receptor expression and calcium flux following anti‐CD3 and anti‐CD2 antibody‐triggered T cell activation. Antigen (tetanus toxoid, Candida and allogeneic cell)‐induced proliferation was detectable. In contrast, cytotoxic T cell activity towards allogeneic cells was deficient. These findings shed light on the function of the TcR/CD3 complex and indicate that the expression of a limited number of TcR/CD3 receptors may be sufficient to trigger antigen‐specific T cell activation (and, possibly, differentiation) and that anti‐CD3 antibody‐induced T cell activation differs somewhat from antigen/major histocompatibility complex molecule‐induced activation. These results also confirm that the CD2 pathway of T cell activation is CD3 dependent.
Biochemical identification of a direct physical interaction between the CD4: p56lck and Ti(TcR)/CD3 complexes
Authors: Burgess Kristine E. , Odysseos Andreani D. , Zalvan Craig , Druker Brian J. , Anderson Paul , Schlossman Stuart F. , Rudde Christopher E.
Published: March 2007

The CD4 and CD8 antigens function in synergy with the TcR/CD3 complex in the generation of intracellular signals leading to T cell proliferation. The association of the protein‐tyrosine kinase p56lck with CD4 and CD8 provides a potential mechanism in the generation of intracellular signals. Several studies have shown that CD4 can co‐modulate with TcR/CD3 suggesting that these receptor complexes may associate on the surface of the T cell. Nevertheless, it has proven difficult to formally demonstrate a direct physical interaction between the CD4 and TcR/CD3 complexes using biochemical techniques. In this study, we have used the sensitivity of the in vitro kinase assay to show a direct physical linkage between the CD4: p56lck complex and various CD3 subunits. Immunoprecipitation of CD4 from cell lysates derived from the T lymphoblastoid line HPB‐ALL results in the co‐purification of p56lck with an additional polypeptide at 20 kDa. Re‐precipitation analysis and isoelectric focusing demonstrated that this band corresponds to the CD3ϵ chain. An alternative approach which involves the labeling of microsomal membranes with [γ−32P]ATP revealed the presence of CD3ϵ and ζ chains in anti‐CD4 immunoprecipitates. By contrast, we were unable to demonstrate the association of the CD4: p56lck and TcR/CD3 complex in resting peripheral blood lymphocytes. These data indicate that the CD4: p56lck and TcR/CD3 complexes have the ability to form stable complexes on the surface of certain T cell lines.
Effect of aging on the autoimmune response to rat male accessory glands: deficit of I‐E‐positive peritoneal cells capable of inducing suppression
Authors: Pistoresi‐Palencia Maria C. , Romero‐Piffiguer Marta , Ferro Maria Elena
Published: March 2007

The present report analyzes the ability to induce suppression to rat male accessory gland (RAG) autoantigens and the characteristics of T suppressor (Ts)‐inducer peritoneal cells (PC) in old rats which show increased autoimmune responses. The injection of young rats with a purified fraction (FI) of RAG 10 and 3 days prior to immunization with chemically modified RAG (MRAG) markedly reduced the immune response to RAG autoantigens when compared with young rats which had only been immunized (controls), while the pretreatment of old rats did not block the delayed‐type hypersensitivity reaction to MRAG when compared with control old rats.

The study of cell surface markers on PC from rats injected i.p. 2 h previously with FI of RAG (FI‐PC) showed an increase of OX‐6 (I‐A) and a decrease of OX‐17 (I‐E) in FI‐PC of old rats with respect to FI‐PC of young animals, which showed a selective increase of I‐E+ Ts‐inducer PC. The i.p. injection of FI‐PC from old rats into young recipients, 10 and 3 days prior to immunization with MRAG in complete Freund's adjuvant, did not modify the autoimmune response when compared with controls. By contrast, the injection of young and old rats with FI‐PC from young animals induced a significant suppression of the autoimmune response.

The reduced percentage of I‐E+ suppressor‐inducer PC provides an explanation for the diminished ability to induce suppression to RAG autoantigens in old rats.
Redistribution of critical major histocompatibility complex and T cell receptor‐binding functions of residues in an antigenic sequence after biterminal substitution
Authors: Reddehase Matthias J. , Koszinowski Ulrich H.
Published: March 2007

Residues critical for establishing a trimolecular interaction with a major histocompatibility complex (MHC)‐encoded receptor and a T cell antigen receptor (TcR) were determined for an antigenic nonapeptide. The N‐terminal residue proved to be involved in binding of the peptide to both receptors and the C‐terminal residue was essential for MHC binding. While substitution of either of these critical terminal residues by alanine resulted in an almost complete loss of peptide antigenicity, simultaneous substitution of both created a new functional ligand for the same MHC molecule and the same TcR. Notably, in the biterminally substituted peptide, the core residues took on new roles in the trimolecular interaction in that a residue critical in the authentic nonapeptide for TcR binding became critical for MHC binding and former spacer residues became essential to various degrees for the interaction with either receptor or both. Thus, apparently, the loss of the terminal residues' contribution was at least partially compensated by a redistribution of the roles among the remaining residues. These results reflect a cooperative contribution of all residues of an antigenic peptide to its binding to both receptors and thus challenge a static definition of agretope and epitope as MHC and TcR binding sites.
Interleukin 4 induces CD8α expression on T cell receptor Vγ5 thymocytes
Authors: Leclercq Georges , De Smedt Magda , Plum Jean
Published: March 2007

It is generally accepted that most T cell receptor (TcR) γ/δ cells are CD4−CD8−. After in vitro culture; however, a low percentage of these cells express the CD8 α subunit. We show here that addition of recombinant interleukin (IL) 4 to IL 2‐cultured murine TcR Vγ5 thymocytes induces the expression of CD8α; CD8β is not expressed. Co‐addition of the anti‐IL 4 mAb 11B11 abrogates the induction of CD8 α expression, ruling out the possibility of a contaminant. Furthermore, we demonstrate that a substantial part of freshly prepared TcR Vγ5 thymocytes express CD8 α.
The function of human NK cells is enhanced by β‐glucan, a ligand of CR3 (CD11b/CD18)
Authors: Di Renzo Livia , Yefenof Eitan , Klein Eva
Published: March 2007

Cells responsible for the natural killer (NK) effect in the human blood can be collected in the low‐density lymphocyte subset and the majority of them express CR3. In addition to the iC3b binding site the CR3 molecules possess an epitope which binds β‐glucan. Ligands of this site can deliver activation signals to CR3‐carrying monocytes and neutrophils. We found that the function of NK cells was also potentiated by preincubation with β‐glucan. The treatment increased the proportion of target‐binding lymphocytes and of the damaged target cells in the conjugates. The monoclonal antibody OKM‐1, directed to the β‐glucan‐binding site of CR3, abrogated this effect. Another CR3‐reactive monoclonal antibody, M522, known to activate monocytes and neutrophils, enhanced the NK function.
Dissociation between syncytia formation and HIV spreading. Suppression of syncytia formation does not necessarily reflect inhibition of HIV infection
Authors: Pantaleo Giuseppe , Poli Guido , Butini Luca , Fox Cecil , Dayton Andrew I. , Fauci Anthony S.
Published: March 2007

In this study, we have observed that a disociation may occur in vitro between syncytia formation and HIV spreading‐Efficient HIV spreading and virus replication occurred either in HIV‐infected LFA‐1+ lymphocytes treated with anti‐LFA‐1 mAb or in HIV‐infected lymphocytes genetically deficient in LFa‐1, despite the fact that syncytia formation was completely suppressed. Therefore, these results indicate that syncytia formation cannot be used as the sole parameter to evaluate the spread of HIV in vitro.
Generation of long‐lived B cells in germ‐free mice
Authors: Förster Irmgard , Müller Werner , Schittek Birgit , Rajewsky Klaus
Published: March 2007

In the present study we have addressed the question of whether the composition of the peripheral B cell pool is similar in mice raised under conventional, specific pathogen‐free or germ‐free breeding conditions. For this purpose, the representation of various B lineage subsets was quantitated by flow cytometry and the lifespan of B cells analyzed by labeling dividing cells with the thymidine analogue 5‐bromo‐2′‐deoxyuridine in vivo. The results indicate that except for an increased fraction of CD45R(B220)dull B lineage cells in the bone marrow and a marginally faster turnover of splenic B cells in germ‐free compared to conventional mice, there is no obvious difference in the representation and turnover of B cells in the different kinds of mice. The data indicate that a stable pool of peripheral B cells is generated even in the absence of microbial stimulation.
Framework peptides from xIIIb rheumatoid factor light chains with binding activity for aggregated IgG
Authors: Hay Frank Charles , Soltys Andzriej Jan , Tribbick Gordon , Geysen H. Mario
Published: March 2007

Most monoclonal human rheumatoid factors (RF) and some RF from rheumatoid patient's synovia are restricted in their light chains, using predominantly the xIIIb subfamily. Very few sequence differences are found between these light chains. Light chains with similar variable region framework sequences are also found in some mouse monoclonal RF derived from mice stimulated with lipopolysaccharide or secondarily immunized with protein antigens.

There are two likely explanations for this restriction in framework sequences between the two species: (a) the sequences are important for the immunoregulation of RF production or (b) the sequences are concerned with the antibody binding specificity of the RF. We have examined overlapping octapeptides from the xIIIb light chain variable region and show that some framework peptides have the ability to bind aggregated IgG. Replacement of amino acids within the peak binding peptide have indicated the critical amino acids necessary for binding.
Components of an antigen‐/T cell receptor‐independent pathway of lymphokine production
Authors: Germann Tieno , Jin Shen‐Chu , Mattner Frank , Rüde Erwin
Published: March 2007

The general way to induce the synthesis of lymphokines by T cells is the stimulation through the T cell receptor (TcR) complex which results in an increase of intracellular [Ca2+] and in the activation of a tyrosine kinase as well as of protein kinase C. Lymphokine production induced via the TcR is inhibited by the immunosuppressive drug eyclosporin A (CsA). However, an alternative pathway of lymphokine production exists. Several T lymphocyte clones can synthesize interferon‐γ (IFN‐γ), granulocyte‐monocyte colony‐stimulating factor, and small amounts of interleukin (IL 3) when stimulated with syngeneic or allogeneic accessory cells (AC) plus IL 2. In contrast to the TcR pathway the alternative pathway does not require a rise of intracellular [Ca2+] and is insensitive to the effects of CsA. In this report we provide evidence for the involvement of T cell‐stimulating factor (TSF) ‐ a probably novel murine cytokine ‐ in the alternative pathway of lymphokine production. It is shown that fixation of the AC with carbodiimide or treatment of the AC with UV light greatly reduces their capacity to induce (in combination with IL 2) the synthesis of IFN‐γ by T cells. This function is restored by addition of TSF. Moreover, TSF alone (without IL 2) in combination with fixed AC can induce the synthesis of substantial amounts of IFN‐γ. Furthermore, TSF in combination with IL 2 can stimulate freshly isolated spleen cells to produce IFN‐γ. The target cell resides probably in the non‐B cell, non‐T cell population.
Production of interleukin 2 and interleukin 4 by immune CD4−CD8+ and their role in the generation of antigen‐specific cytotoxic T cells
Authors: Horvat Branka , Loukides Jill A. , Anandan Linda , Brewer Erin , Flood Patrick M.
Published: March 2007

In this report we investigated the production and role of interleukin (IL)2 and IL4 in the generation of antigen‐specific cytotoxic T cells (CTL). We used as our model the ultraviolet‐light‐induced epithelial tumor 1591, a highly immunogenic regressor tumor which evokes a strong cell‐mediated immune response leading to rejection. We show that IL2 and IL4 are differentially required for the development of optimal cytolytic activity to the 1591 tumor in primary and secondary in vitro splenic cultures. First, anti‐IL2 receptor monoclonal antibody (mAb) significantly decreased specific cytotoxicity in both primary and secondary splenic mixed lymphocyte‐tumor cell culture (MLTC) cultures, but anti‐IL4 mAb inhibited the cytotoxic responses only secondary and not primary cultures. Second, when supernatants from MLTC were tested for lymphokine activity, primary cultures produced only IL2 while secondary cultures produced both IL2 and IL4. Splenic cells were then depleted of CD4+ cells by negative selection, or enriched for CD8+ cells by positive selection, and tested for lymphokine production and requirements. CD8+ cells could not generate significant CTL activity in primary cultures, but could in secondary MLTC. The addition of mAb to either IL2 or IL4 significantly inhibited the generation of CTL by CD8+ cells in these secondary MLTC. CD8+ cells were also found to produce both IL2 and IL4 in secondary MLTC by functional and Northern blot analysis. The production of IL2 and IL4 by CD8+ cells occurs during different phases of culture, with IL2 being produced early (days 1 and 2) and IL4 late (days 3‐5). In addition, the requirement of CD8+ cells for both IL2 and IL4 is unique for that lymphokine. These results suggest that both IL2 and IL4 are both produced and required by CD8+ cells during secondary MLTC, and suggest an additional cellular source of IL4 production besides CD4+ T cells during antigen‐specific CTL responses.
Isolation of normal human follicular dendritic cells and CD4‐independent in vitro infection by human immunodeficiency virus (HIV‐1)
Authors: Stahmer Ingrid , Zimmer J. Pascal , Ernst Martin , Fenner Thomas , Finnern Ricarda , Schmitz Herbert , Flad Hans‐D. , Gerdes Johannes
Published: March 2007

Immunohistological and electron microscopy studies of lymph nodes from patients infected with the human immunodeficiency virus 1 (HIV‐1) demonstrated that follicular dendritic cells (FDC), the antigen‐presenting cells of the B cell system, contain and may produce the virus. To elucidate the mode of infection of FDC with HIV‐1 in vitro we developed an improved method for the preparation of single‐cell suspensions of viable FDC with high purity (>90% FDC). These isolated FDC were subjected to human T cell leukemia virus IIIB infection, which was monitored after 4 days in culture using the polymerase chain reaction. We were able to demonstrate that normal human FDC are highly susceptible to infection by HIV‐1. Inhibition experiments with the monoclonal antibody OKT4a demonstrate that this infection is independent of the CD4 molecule.
The human interferon‐γ gene contains an inducible promoter that can be transactivated by tax I and II
Authors: Brown David A. , Nelson Forrest B. , Reinherz Ellis L. , Diamond Don J.
Published: March 2007

T cells infected with the HTLV (human T cell leukemia virus)‐I and ‐II retroviruses often express the lymphokine interferon‐γ (IFN‐γ). We sought the molecular explanation for this phenomena by co‐transfecting the transactivator genes of these human retroviruses together with 5′ flanking sequences of the human IFN‐γ gene linked to a heterologous reporter gene. In the presence of phytohemagglutinin or anti‐T cell receptor monoclonal antibodies and phorbol 12‐myristate 13‐acetate, the IFN‐γ promoter is activated in human T cells, which can be further enhanced by co‐transfection of the tax I or II genes. Using Bal‐31 deletion analysis, we have found that induction by mitogens can be mapped to a region between −284 to −260 (bp) 5′ of the transcriptional start site, which is separable from the sequence(s) further upstream which transmit the synergistic transactivation by tax I or II.
Differential regulation of lymphokine production in mitogen‐stimulated murine spleen cells
Authors: Sander Birgitta , Cardell Susanna , Möller Göran , Möller Erna
Published: March 2007

Activation of murine spleen cells in vitro with soluble anti‐CD3 monoclonal antibody and phorbol 12‐myristate 13‐acetate (PMA) induced an initial production of interleukin 2 (IL2), interferon‐γ (IFN‐γ), IL4 and IL5, followed by a refractory state during which the T cells did not produce lymphokines when stimulated with some common mitogens. The refractory state was long‐Jasting, depended on the presence of anti‐CD3 and PMA but could be reverted by incubation in fresh medium. Pokeweed mitogen (PWM) differed from other mitogens tested, since stimulation by PWM and PMA induced lymphokine production and proliferation also in the refractory cells. Furthermore, PWM stimulation selectively induced IL4 and IFN‐γ production but not IL2 and IL5, as detected by intracellular cytokine‐specific immunofluorescence and in situ hybridization for mRNA. The results indicate differential regulation of lymphokine production in primary lymphocytes.
Germinal center cells express bcl‐2 protein after activation by signals which prevent their entry into apoptosis
Authors: Liu Yong‐Jun , Mason David Y. , Johnson Gerald D. , Abbot Sandra , Gregory Christopher D. , Hardie Deborah L. , Gordon John , MacLennan Ian C. M.
Published: March 2007

B cells undergo selection within germinal centers on the basis of their capacity to be activated by antigen held on follicular dendritic cells. Isolated germinal center B cells in culture kill themselves by apoptosis but this is prevented if their receptors for antigen are cross‐linked. In this study it is confirmed that almost all germinal center B cells, unlike other B cells, do not express the 25‐kDa protein encoded by the bcl‐2 oncogene. Cross‐linking the surface Ig of isolated germinal center cells causes them to express bcl‐2 protein. Two other stimuli which inhibit the entry of germinal center cells to apoptosis result in the expression of bcl‐2 protein. These stimuli are: (a) CD40 antibody and (b) recombinant 25‐kDa fragment of the CD23 protein plus recombinant interleukin 1α. Respectively, these induce germinal center cells to differentiate to resting B cells or plasmablasts. Dual‐fluorescence studies on small lymphocytes confirm the presence of bcl‐2 protein in mitochondria but show that this is also present in other extra‐nuclear areas.

Burkitt lymphoma cells have a phenotype which indicates that they are neoplastic cells of germinal center origin. The expression of bcl‐2 protein by Burkitt lymphoma lines was also studied. Burkitt lines which retain the phenotype of fresh Burkitt lymphoma cells can be induced to enter apoptosis on culture with the Ca2+ ionophore ionomycin. These cells were found not to express bcl‐2 protein. By contrast, Burkitt lines which have drifted towards a lymphoblastoid cell line phenotype and are resistant to the induction of apoptosis express high levels of the bcl‐2 protein.

The findings support the concept that the susceptibility of germinal center cells to entering apoptosis is associated with their lack of expression of bcl‐2 protein. Aberrant expression of bcl‐2 protein by some neoplastic germinal center cells may allow survival in situations where their normal counterparts die.
A single amino acid distinguishes the high‐responder from the low‐responder form of Fc receptor II on human monocytes
Authors: Clark Marcus R. , Stuart Susan G. , Kimberly Robert P. , Ory Peter A. , Goldstein Ira M.
Published: March 2007

The low‐affinity Fc receptor on human peripheral blood monocytes (FcγRIIA) is polymorphic with respect to its ability to bind murine IgG1. The two allelic forms of the receptor, high responder (HR) and low responder (LR), yield characteristic patterns after isoelectric focusing and react differently with the anti‐FcγRII monoclonal antibody (mAb), 41H16. We recently cloned cDNA encoding the extracellular domains of FcγRIIA on monocytes from one HR and two LR donors, and found that they differed at only a single base. The cDNA isolated from the HR donor had a G at position 519 and would be expected to encode an aginine at residue 133 in the mature protein, while the cDNA isolated from both LR donors had an A at position 519 and would be expected to encode a histidine at the same residue. To determine whether this single amino acid substitution actually accounts for the functional polymorphism involving FcγRIIA, we transfected COS cells with full‐length HR and LR FcγRIIA cDNA, and examined them for their ability to react with anti‐FcγRIIA mAb and to bind red blood cells (RBC) coated with either murine IgG2b or murine IgG1. Whereas COS cells transfected with either the HR cDNA or the LR cDNA reacted with the anti‐FcγRII mAb, IV.3, and bound murine IgG2b‐coated RBC, only COS cells transfected with the HR cDNA formed rosettes with murine IgG1‐coated RBC and reacted strongly with mAb 41H16. A total of nine LR donors were identified, and all were homozygous for the A substitution at position 519. We conclude that at an A at position 519 in the cDNA encoding FcγRIIA is the primary molecular basis for the LR form of the receptor, and that the amino acid at residue 133 determines whether FcγRIIA efficiently binds murine IgG1.