bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effect of anti‐interleukin (IL)‐6 antibody against endotoxin, associated with paradoxically increased IL‐6 levels
Authors: Heremans Hubertine , Dillen Chris , Put Willy , Van Damme Jo , Billiau Alfons
Published: March 2007

Two rat monoclonal antibodies (6B4 and 20F3) against mouse interleukin (IL)‐6 were studied for their effects on the generalized Shwartzman reaction and on cytokine production elicited by endotoxin injections. Both antibodies were found to protect mice against the generalized Shwartzman reaction. Production of interferon and tumor necrosis factor in these animals, as assessed from serum levels, were not consistently affected by the antibody treatment, although rather increased levels were occasionally noted. Paradoxically, however, endotoxin‐induced serum levels of IL‐6 in anti‐IL‐6‐treated mice were consistently found to be markedly increased and also to persist for longer time periods. The more vigorous and persistent response may have been due to slower elimination, increased synthesis, or a combination of both. Endogenous production of IL‐6 in mice may be sufficiently large to supersede the neutralizing potential of an excess of antibody, as was evident from the fact that ascites fluid of the anti‐IL‐6‐producing 6B4 hybridoma was biologically active in the IL‐6 assay.
Distribution of interferon‐γ receptor in human tissues
Authors: Valente Guido , Ozmen Laurence , Novelli Francesco , Geuna Massimo , Palestro Giorgio , Forni Guido , Garotta Gianni
Published: March 2007

Interferon‐γ (IFN‐γ) is produced by activated T lymphocytes and plays a regulatory role in immune responses. The nature and location of cells that express the IFN‐γ receptor (R) and respond to this lymphokine are not well documented. The distribution of human IFN‐γ‐R (HuIFN‐γ‐R) was, therefore, investigated in situ by immunohistochemistry, using affinity‐purified rabbit polyclonal antibodies directed against the extracellular domain of the receptor. In lymphoid organs, IFN‐γ‐R expression is restricted to the B cell areas of lymph nodes, adult and fetal spleen, tonsils, appendix, and mucosa‐associated lymphoid tissue of the small bowel. Macrophages and other reticular cells in lymphoid tissues and other organs are strongly positive for IFN‐γ‐R, whereas its expression was consistently negative in the cortical and medullary thymocytes. Two‐color flow cytofluorometric analysis of blood, lymph node, tonsil, spleen and thymus cells confirms that most B lymphocytes are IFN‐γ‐R positive, whereas T lymphocytes are negative. However, after in vitro activation, peripheral blood T cells become IFN‐γ‐R+. In non‐lymphoid organs, IFN‐γ‐R is expressed on endothelial cells of the medium‐ and small‐size vessels. In epithelial tissues, high expression of IFN‐γ‐R is detected on trophoblastic epithelium, glandular cells of stomach, ileum and colon, lung alveolar cells, salivary duct cells, renal tubular cells, and endometrial mucosa cells. Hepatocytes are weakly positive, while squamous epithelial cells are negative. The distribution of the HuIFN‐γ‐R is discussed in view of the known functions of IFN‐γ.
Limited heterogeneity of T cell receptor variable region gene usage in juvenile rheumatoid arthritis synovial T cells
Authors: Sioud Mouldy , Kjeldsen‐Kragh Jens , Suleyman Sel , Vinje Odd , Natvig Jacob B. , Førre Øystein
Published: March 2007

The aim of this study was to determine whether synovial fluid (SF) T cells in patients with juvenile rheumatoid arthritis (JRA) are restricted in their T cell receptor (TcR) gene repertoire. The quantitative polymerase chain reaction (QPCR) was used to compare the transcription of Vβ and Vα gene families in freshly isolated SF T cells, in interleukin‐2 receptor‐positive (IL‐2R+) T cells and in peripheral blood (PB) T cells from 18 patients. Significantly less Vβ families are detected in SF when compared with PB (p > 0.0003). The TcR Vβ gene usage by IL‐2R+ T cells was even less heterogeneous when compared with freshly isolated SF T cells (p > 0.0002). Freshly isolated SF T cells from the left and the right knees of four patients transcribed the same Vβ families. Furthermore, we demonstrate that in SF the distribution of certain TcR Vβ gene segments in CD4+ and CD8+ T cells differed from that in PB of the same patient.

The TcR Vα usage was studied in IL‐2R+ T cells from six patients who had shown restriction in their SF TcR Vβ gene usage. Only two to five TcR α transcripts were detected in three of these patients while a broad TcR Vα usage was seen in the other three patients. Sequence analysis of the SF Vβ 20 cDNA clones generated from the IL‐2R+ T cells of two patients demonstrated an oligoclonal expansion. Taken together, our data could indicate an antigen‐ and/or superantigen‐driven expansion of selected T cells in the synovial compartment.
Restricted diversity of Vγ9‐JP rearrangements in unstimulated human γ/δ T lymphocytes
Authors: Delfau Marie‐Hélène , Hance Allan J. , Lecossier Denise , Vilmer Etienne , Grandchamp Bernard
Published: March 2007

The diversity of human peripheral blood γ/δ T cells is known to be limited by the preferential use of V genes coding for Vγ9 (usually linked to JP) and Vδ2. We show that the diversity of these cells is further limited at the junctional region. First, an identical rearrangement is found in 10%–30% of all γ/δ T cells which contain Vγ9‐JP rearrangements. Second, the vast majority of Vγ9‐JP rearrangements which are different from this predominant sequence have, nevertheless, the same length or code for variable regions whose length differs by only one amino acid (±1). Overall, 30%–50% of Vγ9‐JP rearrangements have a junctional region which encodes for a peptide with the amino acid sequence E VX EL, in which EV is predominantly, but not exclusively, encoded by the germ‐line Vγ9 sequence and EL is encoded by JP. The X amino acid is variable, but a glutamine is over‐represented. The diversity of the Vγ9‐JP repertoire is fairly constant in different individuals and at different ages, including before, during and after the post‐natal expansion of peripheral blood γ/δ T cells.
Passive transfer of experimental myasthenia gravis via antigenic modulation of acetylcholine receptor
Authors: Loutrari Heleni , Kokla Anna , Tzartos Socrates J.
Published: March 2007

Antigenic modulation of acetylcholine receptor (AChR) is considered to contribute to the reduction of endplate AChR in myasthenia gravis (MG). Yet, the pathogenic significance of this mechanism is unclear. To investigate the in vivo role of AChR antigenic modulation we examined the ability of bivalent F(ab')2 and monovalent Fab fragments of monoclonal antibody (mAb) 35 to passively transfer experimental autoimmune MG (EAMG) in rats. mAb 35 which binds at the main immunogenic region (MIR) of the AChR causes severe EAMG without being involved in channel function. Compared to the intact mAb, F(ab')2 35 proved to be less potent but still capable of inducing moderate EAMG, whereas Fab 35 were totally ineffective. Furthermore, both intact and F(ab')2 35 induced mild EAMG in complement‐depleted rats. These results (a) provide evidence that antigenic modulation of endplate AChR is sufficient to generate passive transfer of EAMG and (b) further support the pathogenic potential of the anti‐MIR antibodies in MG.
Peptide motifs of closely related HLA class I molecules encompass substantial differences
Authors: Rötzschke Olaf , Falk Kirsten , Stevanović Stefan , Jung Günther , Rammensee Hans‐Georg
Published: March 2007

The peptides presented by major histocompatibility complex class I molecules adhere to strict rules concerning peptide length and occupancy by certain amino acid residues at anchor positions. Peptides presented by HLA‐A*0201 molecules, for example, are generally nonapeptides requiring Leu or Met at position 2 and an aliphatic residue, predominantly Val, at position 9. A closely related molecule, HLA‐A*0205, differing from the former at four amino acid residues, has a related but substantially different peptide motif. A*0205‐presented peptides are still nonapeptides, and position 9 is still aliphatic, although it is preferentially occupied by Leu instead of Val. Position 2 not only allows aliphatic residues but also polar ones. Occupancy at position 6, considered as an auxiliary anchor in A*0201, as well as non‐anchor residues at positions 3, 4, and 8 are relatively well conserved between the two peptide motifs. Thus, although a number of the T cell epitopes presented by the two HLA‐A2 forms is expected to be identical, a considerable number of epitopes should be different.
A new subpopulation of intestinal intraepithelial lymphocytes expressing high level of T cell receptor γδ
Authors: Tauchi Yoshihiko , Matsuzaki Goro , Takimoto Hiroaki , Yoshikai Yasunobu , Nomoto Kikuo
Published: March 2007

The murine intestinal epithelium contains T cell receptor (TcR) γδ‐bearing T cells in high frequency. In the present report, we showed that TcR γδ‐bearing intestinal intraepithelial lymphocytes (IEL) from C3H/He (H‐2k) mice can be divided into two subpopulations based on TcR expression level; a subpopulation with a remarkably high level of TcR expression and a subpopulation with a moderate level of TcR expression, designated as TcR γδhi IEL and TcR γδmod IEL, respectively. In flow cytometric analysis, the TcR γδhi IEL expressed a high level of TcR (mean fluorescence channel 518) when compared with the TcR level of TcR γδ+ T cells in other lymphoid organs (mean fluorescence channel 88). The TcR γδhi IEL were detected in IEL from mice of H‐2k and H‐2k/b haplotypes but not in H‐2b and H‐2d haplotypes. Vδ4, which was reported to be frequently expressed in IEL of H‐2k mice, was preferentially expressed by TcR γδhi IEL. These results suggested that the existence of the TcR γδhi population is related to the high frequency of Vδ4 in H‐2k mice.
Why is clonal deletion of neonatal thymocytes defective?
Authors: Signorelli Kathy , Benoist Christophe , Mathis Diane
Published: March 2007

A major mechanism for establishing tolerance to some murine self antigens is clonal deletion of self reactive T cells in the thymus. This mechanism is responsible for the near absence of T cells displaying particular T cell receptor (TcR) Vβ in strains of mice that express the major histocompatibility complex class II E molecule and a protein encoded within the 3′ open reading frame (ORF) of certain endogenous mammary tumor viruses (Mtv). However, clonal deletion does not operate in these same strains during the first few days after birth. This defect could be explained by a difference in any (or any combination of) the three elements involved: the T cell, the thymic stromal cell(s) or the antigen. We have explored these different possibilities and have come to the conclusion that a lack of antigen is the most likely explanation. Yet, neonatal and adult thymi have quite similar levels of messenger ribonucleic acid corresponding to Mtv 3′ ORF.
Membrane region of surface IgM is not sufficient for transducing growth inhibitory signals in an immature B cell line WEHI‐231
Authors: Iwabuchi Naoto , Williams Douglas B. , Nguyen Hai P. , Wu Yongjian , Tisch Roland , Azuma Toshifumi , James Phillips M. , Hozumi Nobumichi
Published: March 2007

The murine B lymphoma line WEHI‐231 is representative of immature B cells. Like normal immature B cells, WEHI‐231 is susceptible to growth arrest following cross‐linking of surface IgM (sIgM). Previously, we have shown using a WEHI‐231 immunoglobulin (Ig) δ‐transfectant that sIgD cross‐linking failed to initiate growth arrest, in contrast to sIgM. In this report, we extend our research to investigate the structural requirement of Ig μ chain for regulating growth inhibition. Recombinant, chimeric Ig molecules δ/μm and μ/δm consisting of exons encoding extracellular δ and μ domains and membrane regions of different isotypes were constructed and introduced into WEHI‐231 cells. A similar approach was used for sIgG2b‐expressing transfectants. Our findings indicate that the μm region is not sufficient for regulation of growth inhibition in WEHI‐231 cells and suggest that additional extracellular region(s) of μ chain may be required for this response.
Correlation of calcineurin phosphatase activity and programmed cell death in murine T cell hybridomas
Authors: Fruman David A. , Mather Pamela E. , Burakoff Steven J. , Bierer Barbara E.
Published: March 2007

Ligation of T cell receptor/CD3 complexes induces programmed cell death, or apoptosis, in immature thymocytes and many T cell hybridomas. While it has been demonstrated that T cell receptor‐mediated apoptosis requires an increase in intracellular calcium concentration, the specific calcium‐dependent signalling events leading to cell death are poorly defined. We have previously shown that T cell receptor/CD3‐mediated induction of apoptosis in a murine T cell hybridoma is inhibited by the immunosuppressive drugs cyclosporin A (CsA) and FK506. Recently, it has been determined that these agents inhibit the activity of calcineurin, a calcium‐ and calmodulin‐dependent serine/threonine phosphatase. Using an assay which measures calcineurin activity in cell lysates, we find that calcineurin‐dependent dephosphorylation of a phosphopeptide substrate is potently inhibited in hybridomas treated with CsA or FK506. Drug dose‐response analyses indicate that the level of cellular calcineurin activity correlates closely with the ability of these cells to undergo apoptosis. Thus, calcineurin appears to be a critical mediator of T cell receptor/CD3 signalling leading to programmed cell death in T cell hybridomas.
Evidence for endocytosis of E‐selectin in human endothelial cells
Authors: Von Asmuth Eckhardt J. U. , Smeets Edgar F. , Ginsel Leo A. , Onderwater Jos J. M. , Leeuwenberg Jet F. M. , Buurman Wim A.
Published: March 2007

E‐selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)‐activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule‐1 (ICAM‐1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E‐selectin, was internalized at a rate of approximately 1.7% of the membranebound [125I]mAb per minute. In contrast, less than 0.1% of membrane‐bound [125I]RR1/1, an mAb specific for ICAM‐1, was internalized per minute. TNF‐activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E‐selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid‐phase internalization, were detected in the same organelles, thus arguing against mAb interaction‐induced E‐selectin internalization. Furthermore, the amount of E‐selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E‐selectin kinetics.

Taken together, these results indicate that TNF‐activated HUVEC constitutively internalize E‐selectin. Physiological significance of E‐selectin internalization in the regulation of E‐selectin membrane expression, and in clearing E‐selectin ligands from the circulation, needs further investigation.
Neurons and neuroblastoma as a source of macrophage colony‐stimulating factor
Authors: Nohava Kathrin , Malipiero Ursula , Frei Karl , Fontana Adriano
Published: March 2007

Accumulation of macrophages in brain tissue as observed in nervous system injury may be due to local production of hematopoietic colony‐stimulating factors (CSF). The present work shows human neuroblastoma cells and murine neurons, namely granule cells of the cerebellum, to produce macrophage (M)‐CSF which guides expansion and differentiation of macrophage lineage cells. The mRNA‐encoding M‐CSF but not the respective protein is present in mouse brain including cerebellum. Neither granulocyte M‐CSF nor IL‐3 is produced by cerebellar neurons or neuroblastoma. By their production of M‐CSF, neurons may regulate the macrophage response and lead to local expansion and enhanced function of macrophages in inflammatory diseases of the central nervous system.
Tyrosine kinase activation in thymic epithelial cells: necessity of thymocyte contact through the gp23/45/90 adhesion complex
Authors: Coutrure Clément , Amarante‐Mendes Gustavo , Potworowski Edouard F.
Published: March 2007

Interactions between thymocytes and thymic stromal cells are necessary for T cell differentiation, maturation and proliferation. The signals required for these events to occur often necessitate close contact, and indeed adhesion, between the cell types involved. While the transmission of signals from stromal cells to thymocytes has been well documented, there is little evidence that binding of thymocytes to stromal cells can result in stromal cell activation. We have recently identified a novel thymic epithelial adhesion complex composed of three non‐covalently associated glycoproteins (gp23, gp45 and gp90). While gp23 and gp45 are jointly required for adhesion to thymocytes, the function of gp90 is unknown. In the present work, we show that gp23/45‐mediated contact with thymocytes induces de novo tyrosine phosphorylation of gp90. Furthermore, the protein tyrosine kinase responsible for gp90 neophosphorylation is itself an integral part of the adhesion complex.
Analysis of human/mouse interleukin‐6 hybrid proteins: both amino and carboxy termini of human interleukin‐6 are required for in vitro receptor binding
Authors: Fiorillo Maria Teresa , Cabibbo Andrea , Iacopetti Paola , Fattori Elena , Ciliberto Gennaro
Published: March 2007

The multifunctional cytokine interleukin‐6 (IL‐6) is a single polypeptide chain consisting of 184 amino acids in man and 187 amino acids in mouse. Despite the relatively high degree of sequence similarity of these two molecules (about 57%), the biological activity in mouse and human IL‐6 shows species specificity. Starting with this observation, we constructed interspecies hybrids with the goal of defining which segments of the human IL‐6 molecule are involved in human receptor binding. In this manner we generated multiple amino acid substitution mutants which do not contain insertions or deletions as compared with the parental proteins, and which, therefore, should not show dramatic changes in folding. Using two biological assays on cells of human and mouse origin and a recently developed in vitro binding assay to recombinant soluble human IL‐6 receptor, we obtained results which indicate that both the amino and carboxy termini are necessary and sufficient for efficient binding, but that the carboxy terminus plays the dominant role in receptor recognition.
Molecular detection and in vivo analysis of the specific T cell response to a protein antigen
Authors: Cochet Madeleine , Pannetier Christophe , Regnault Armelle , Darche Sylvie , Leclerc Claude , Kourilsky Philippe
Published: March 2007

We have analyzed in detail the repertoire of transcripts encoding the Vβ chains of the T cell receptor and investigated the T cell response of B10.A mice to pigeon cytochrome c. We were thus able to follow the specific T cell response in vivo after immunization with this protein antigen. The response is first detectable in the draining lymph nodes, then in the spleen and in the blood. It is qualitatively similar in individual animals. It is dominated by a major category of specific T cells harboring a Vβ3‐Jβ1.2 rearrangement, and a limited and well‐defined set of nucleotide sequences, previously found in several specific T cell hybridomas and clones. This predominance is observed from the onset of the immune response strongly suggesting the notion that there is no variation and, therefore, no maturation of the T cell response in the course of immunization.
The CD39 molecule defines distinct cytotoxic subsets within alloactivated human CD8‐positive cells
Authors: Gouttefangeas Cécile , Mansur Indra , Schmid Michel , Dastot Hélène , Gélin Catherine , Mahouy Guy , Boumsell Laurence , Bensussan Armand
Published: March 2007

Lymphocyte activation induces or increases the expression of several surface structures, none of which is characteristic of an activated cell subset. In particular, structures such as CD45RO, CD25, CD26, CD49b, CD54, CD71 are expressed by the vast majority of lymphocytes at various times following in vitro activation. CD39 molecules were originally identified on activated B lymphocytes and have recently been described on activated T cell clones. In the present report, we have characterized phenotypically and functionally defined cell subsets generated during an in vitro allostimulation. Results indicated that the percentage of CD39+ cells reached a maximum at day 6 and remained stable thereafter. We demonstrate that CD39 expression allows the identification within the allosensitized CD8+ cytotoxic cells of distinct subsets of cells mediating allo cytotoxic T lymphocyte or natural killer (NK)‐like reactivity. More precisely, CD8+CD39+ alloactivated cells mainly mediate specific killer activity, whereas CD8+CD39− alloactivated cells predominantly exhibit NK‐like reactivity. Further, we show a high functional correlation associated with the lack of CD39 expression on NK‐like alloactivated CD8+ cells, while there is no association with CD56 or CD57 NK‐associated structures.
Leukotriene B4 transcriptionally activates interleukin‐6 expression involving NK‐xB and NF‐IL6
Authors: Brach Marion A. , De Vos Sven , Arnold Christian , Gruß Hans‐Jürgen , Mertelsmann Roland , Herrmann Friedhelm
Published: March 2007

Leukotriene B4 (LTB4) is a notable participant in inflammation and chemotaxis. It is, however, still unclear whether LTB4 acts in this regard directly or indirectly by stimulating the release of chemotactic and inflammatory cytokines. Here we report that LTB4 induces synthesis of interleukin (IL)‐6 by human blood monocytes through transcriptional activation of the IL‐6 gene. We furthermore demonstrate that this process involves activation of the transcription factor NF‐xB and, to a lesser extent, of NF‐IL6, while the activity of the transcription factor AP‐1, shown to otherwise confer IL‐6 inducibility, appeared to be unaffected by LTB4. Involvement of NF‐xB and NF‐IL6 in induction of IL‐6 transcription by monocytes was demonstrated using deleted forms of the IL‐6 promoter. Activation of the IL‐6 promoter by LTB4 was not only associated with accumulation of the respective transcripts but resulted in synthesis of functional IL‐6 protein as well. In addition, LTB4 mediated transactivation of a heterologous promoter construct containing the NF‐xB or the NF‐IL6 enhancer, but not the AP‐1 enhancer. The signaling events mediating this effect appeared to involve the release of H2O2, since LTB4 failed to induce NF‐xB or NF‐IL6 in the presence of the scavenger of H2O2, N‐acetyl‐L‐cysteine.
Effects of HIV‐1 Tat protein on human T cell proliferation
Authors: Meyaard Linde , Otto Sigrid A. , Schuitemaker Hanneke , Miedema Frank
Published: March 2007

In inducing immunodeficiency in human immunodeficiency virus (HIV)‐1 infection, a role for the HIV‐1 Tat protein has been suggested. In addition to effects on viral transcription, the protein has been ascribed immunosuppressive functions, which were investigated in this study. Proliferation of purified T cells to CD3 monoclonal antibodies immobilized to plastic plates was inhibited up to 70% by addition of 5 μg/ml of the Tat protein. This inhibitory effect, however, was not observed in the presence of accessory cells. Furthermore, no effect of Tat protein could be observed on T cell proliferative responses to recall antigen which most likely is related to the presence of accessory cells in the cultures. Taken together, our results do not imply an important role for immunosuppressive effects of Tat in induction of immunodeficiency as observed in HIV infection in vivo.
Nitric oxide mediates suppression of T cell responses in murine Trypanosoma brucei infection
Authors: Sternberg Jeremy , McGuigan Fiona
Published: March 2007

African trypanosomes induce a generalized state of immunosuppression in their mammalian hosts. One characteristic of this is a suppression of lymphocyte responses to mitogen, which is mediated by suppressor macrophages. We investigated the involvement of nitric oxide in this phenomenon. Both peritoneal and splenic cell cultures from infected mice released nitrite and this was inhibitable by NG‐monomethyl L‐arginine (L‐NMMA). The release of nitrite correlated with suppressed splenic T cell proliferative responses to concanavalin A. It was shown that adherent spleen cells from infected mice mediate suppression, which could be abrogated by L‐NMMA. These results suggest that in T. brucei infection, the activation of macrophages to produce nitric oxide leads to impaired lymphocyte responses and immunosuppression.
Quantitative analysis of the response of human T cell receptor Vγ9+ cells to Plasmodium falciparum
Authors: Goodier Martin , Krause‐Jauer Maria , Langhorne Jean
Published: March 2007

Plasmodium falciparum stimulates peripheral blood γδ+ T cells from unexposed donors. The responding cells bear Vγ9+ chains of the T cell receptor, the majority of which, but not all, are associated with Vγ2 chains. We have analyzed whether the precursor frequency of these Vγ9+ cells approaches that expected of superantigens or mitogens and whether, like a superantigen, the response is major histocompatibility complex (MHC) unrestricted. Limiting dilution analyses of the response of enriched γδ+ T cells to P. falciparum using autologous and heterogolous antigen‐presenting cells suggest that the response is more characteristic of an antigen‐specific MHC‐restricted response. The frequencies are lower than would be expected if all Vγ9+ cells respond, and there is a dramatically reduced response when allogeneic antigen‐presenting cells are used.
High frequency of somatically mutated IgM molecules in the human adult blood B cell repertoire
Authors: Van Es Johan H. , Meyling Frits H. J. Gmeli , Logtenberg Ton
Published: March 2007

Nucleotide sequence analysis of cDNA encoded by the single member of the human immunoglobulin VH6 gene family show that blood B cells in adults, but not in neonates, frequently express somatically mutated IgM molecules. The number of mutations in VH6‐encoded cDNA from adult blood ranged from 2 to 19 mutations/VH gene (average 10.1/VH gene). The distribution of silent and replacement mutations suggests that at least some of the VH6 genes were derived from B cells that were activated and selected by antigen. We conclude that the blood B cell repertoire in adult humans, in contrast to its much‐studied murine splenic counterpart, is a rich source of highly mutated IgM molecules.
Regulation of interleukin‐4 production in human mononuclear cells
Authors: Holter Wolfgang , Majdic Otto , Kalthoff Frank S. , Knapp Walter
Published: March 2007

Interleukin (IL)‐4 is a cytokine with a broad range of effects on immune cells, however, little is known regarding the regulation of its production in freshly isolated human peripheral blood mononuclear cells (PBMC). Here we report the production of IL‐4 in such cells following stimulation with monoclonal antibodies (mAb) directed against different cell surface antigens. We show that triggering via CD2 is more efficient for IL‐4 production than triggering via the CD3 complex. The addition of a CD28 mAb enhances IL‐4 production approximately threefold. Cell depletion experiments show that among CD2 plus CD28‐stimulated PBMC the production of IL‐4 is restricted to the CD8−CD45RA−T cell subpopulation. mAb interfering with the binding of IL‐2 to its receptor can inhibit the production of IL‐4 in CD2 plus CD28‐stimulated PBMC. As IL‐2 induces cell proliferation and production of interferon‐γ, but not production of IL‐4, it follows that IL‐2 is necessary but not sufficient for IL‐4 production.
The viral superantigen Mls‐1a induces interferon‐γ secretion by specifically primed CD8+ cells but fails to trigger cytotoxicity
Authors: Herrmann Thomas , Waanders Gary A. , Chvatchko Yolande , Robson MacDonald H.
Published: March 2007

Superantigens can be operationally defined by their ability to stimulate CD4+ and CD8+ T cells via the T cell receptor β chain variable domain (TcR Vβ). We show here that effector functions of CD8+ T cells specific for superantigens differ depending upon the nature of the superantigen involved. Hence, activated CD8+ T cells bearing TcR Vβ specific for the superantigen Mls‐1a [encoded in the open reading frame of the 3′ long terminal repeat of endogenous mouse mammary tumor virus (MMTV)] are unable to lyse Mls‐1a‐bearing target cells despite the fact that they release interferon‐γ (IFN‐γ) upon Mls‐1a stimulation. In contrast CD8+ T cells specific for the exogenous superantigen staphylococcal enterotoxin B (SEB) readily mediate both lysis and IFN‐γ secretion when exposed to SEB‐bearing target cells. This dissociation between lysis and IFN‐γ production by Mls‐1a‐specific CD8+ T cells is independent of the initial stimulus used for activation and appears not to be simply explained by a low Mls‐1a determinant density. We suggest that this phenomenon reflects differing TcR affinity thresholds for lymphokine secretion and cytolysis. Such differences may be exploited by retroviruses such as MMTV in order to escape immunosurveillance.
Human growth factor for murine interleukin (IL)‐9 responsive T cell lines: co‐induction with IL‐6 in fibroblasts and identification as LIF/HILDA
Authors: Van Damme Jo , Uyttenhove Catherine , Houssiau Frédéric , Put Willy , Proost Paul , Van Snick Jacques
Published: March 2007

Two mouse helper T cell clones that proliferate in response to murine interleukin (IL)‐9 could also be grown in conditioned medium of stimulated human connective tissue cells. The activity was not due to known T cell growth factors including human IL‐9, which is not effective on mouse cells. This growth‐stimulatory activity for TS1 cells (GATS) was co‐induced with IL‐6 on normal fibroblasts and certain sarcoma cell lines stimulated with IL‐1, double‐stranded RNA, virus or phorbol ester. However, the conditions for optimal induction and the kinetics of production were found to be different for IL‐6 and GATS. GATS from phorbol ester‐stimulated human hepatosarcoma cells co‐purified with IL‐6, but could be separated from it by subsequent cation‐exchange fast‐protein liquid chromatography and reverse‐phase high‐performance liquid chromatography. Homogeneous tumor cell‐derived GATS was a 25‐kDa protein on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, whereas IL‐6 produced by these cells appeared in its 23‐kDa form. Pure GATS was found to be inactive in the B cell hybridoma growth assay for IL‐6. Finally, GATS was identified by NH2‐terminal sequence analysis of the mature protein as leukemia inhibitory factor or human interleukin for DA cells (LIF/HILDA). The effect of LIF/HILDA on T cells was not mediated by IL‐2, IL‐4 or IL‐9 production. Since this cytokine has not previously been reported to act on T cells, further investigation of its role in T cell activation should be taken into consideration.
High amounts of circulating interleukin (IL)‐6 in the form of monomeric immune complexes during anti‐IL‐6 therapy. Towards a new methodology for measuring overall cytokine production in human in vivo
Authors: Lu Zhao Yang , Brochier Jean , Wijdenes John , Brailly Hervé , Bataille Régis , Klein Bernard
Published: March 2007

A patient with plasma cell leukemia was treated with anti‐interleukin (IL)‐6 monoclonal antibodies (mAb) for 2 months. Using chromatography on protein A‐Sepharose, anti‐murine‐IgG‐Sepharose, anti‐IL‐6‐mAb‐Sepharose and gel filtration at pH 2.3, we have demonstrated that the anti‐IL‐6 mAb, by preventing the binding of IL‐6 to its cell membrane receptor and its renal elimination, has induced huge amounts of IL‐6 to circulate in the form of monomeric immune complexes. By using this observation, we have developed a mathematical modelling that allows the determination of the overall daily production of IL‐6 in this patient, which was in the range of 15 μg per day. Overall in vivo production of cytokines has never been evaluated in animals or in humans before.
Co‐stimulation of murine CD4 T cell growth: cooperation between B7 and heat‐stable antigen
Authors: Liu Yang , Jones Bryan , Brady William , Janeway Charles A. , Linley Peter S.
Published: March 2007

The B cell activation antigen B7/BB1 has been shown to co‐stimulate growth of human T cells by binding the T cell molecule CD28. In mice, the heat‐stable antigen (HSA) has also been shown to act as a co‐stimulator for T cell growth. In this study, we have evaluated the contributions of B7 and HSA to the co‐stimulatory activity of antigen‐presenting cells (APC). Mouse B7 provides co‐stimulatory activity for murine CD4 T cells in anti‐CD3‐induced proliferation. Human CTLA4Ig, a chimeric molecule comprising the extracellular region of CTLA‐4 fused to an immunoglobulin Cγ fragment, binds to murine B7. We, therefore, use human CTLA4Ig and the hamster anti‐HSA monoclonal antibody 20C9 to analyze the relative contributions of B7 and HSA to the co‐stimulatory activity of murine spleen APC. Our data reveal that both murine B7 and HSA are expressed by dendritic cells and by low‐density spleen B cells. Either CTLA4Ig alone or anti‐HSA alone inhibited CD4 T cell proliferation to anti‐CD3 by > 90%, while CTLA4Ig and anti‐HSA together were far more efficient in inhibiting clonal expansion of CD4 T cells. These results demonstrate that functionally defined co‐stimulation involves at least B7 and HSA and suggest that signals delivered by B7 and HSA synergize in promoting T cell growth.
Interleukin‐4 differentially regulates interleukin‐2‐mediated and CD2‐mediated induction of human lymphokine‐activated killer effectors
Authors: Robinet Eric , Kamoun Malek , Farace Françoise , Chouaib Salem
Published: March 2007

Natural killer (NK) cells can be differentiated into lymphokine‐activated killer (LAK) effectors following stimulation with interleukin (IL)‐2. This induction can be negatively regulated by IL‐4. In this study, we demonstrate that the stimulation of NK cells through the CD2 pathway with (9‐1 + 9.6) monoclonal antibodies can also induce these cells to secrete tumor necrosis factor‐α (TNF‐α) and to differentiate into LAK effectors. More importantly, our data indicate that, in contrast to the IL‐2‐induced LAK generation, the anti‐CD2‐triggered LAK activity was not regulated by IL‐4. IL‐4 was found to enhance the LAK activity as well as NK cell proliferation following activation with anti‐CD2 by a mechanism involving, at least in part, an increased TNF‐α production. Using immobilized monoclonal antibodies against the Fc receptor (FcγRIII or CD16) for NK stimulation, we also observed that the anti‐CD16‐induced LAK activity was not inhibited by IL‐4.

These data further point to a pivotal role of TNF‐α as a regulatory cytokine in anti‐CD2‐induced LAK generation, and suggest that IL‐4 could serve as a discriminatory factor between two distinct pathways involved in the activation of non‐MHC‐restricted cytotoxicity.
Molecular characterization of the Vγ9 T cell receptor repertoire expressed in patients with rheumatoid arthritis
Authors: Olive Colleen , Gatenby Paul A. , Serjeantson Susan W.
Published: March 2007

We have characterized the variable (V)γ9 T cell receptor (TcR) repertoire expressed in synovial membrane (SMC) and peripheral blood mononuclear cells (PBMC) of five rheumatoid arthritis (RA) patients. The experimental approach was to sequence the junctional regions of polymerase chain reaction (PCR)‐amplified Vγ9 transcripts; γ chain sequences were compared with those found in normal PBMC. For normal PBMC, a large proportion of Vγ9 transcripts used the joining (J)γP gene segment whereas a few used Jγ2. Despite this restriction in Jγ usage, there was extensive junctional region diversity with a unique sequence observed in each transcript examined. In contrast to normal PBMC, Jγ usage of Vγ9 transcripts in PBMC of two patients was skewed towards Jγ2. This deviation in Jγ usage was more pronounced in SMC since all patients expressed Vγ9 transcripts in SMC which predominantly used Jγ2, as opposed to JγP in normal PBMC. Further, approximately 60% of Vγ9 transcripts in PBMC of each of three patients had identical junctional region sequences, although the specific sequences were unique in each patient. For two of these patients the dominant transcript found in the PBMC was detected in the corresponding SMC at about 10% and 40%, respectively. Overall, our findings indicate that Vγ9‐bearing T cells in RA peripheral blood are largely derived from clonal expansion whereas in the synovium there is expression of a population of these cells that are mainly polyclonal. This may reflect in vivo expansion in response to a Vγ9/Jγ2 region‐specific antigen. The data presented in this report suggest that Vγ9+ T cells may play a role in the development of RA.
Both T cell receptor (TcR)‐CD3 complex and CD2 increase the tyrosine kinase activity of p56lck. CD2 can mediate TcR‐CD3‐independent and CD45‐dependent activation of p56lck
Authors: Danielian Silvia , Alcover Andrés , Polissard Laurence , Stefanescu Maria , Acuto Oreste , Fischer Siegmund , Fagard Remi
Published: March 2007

T cell activation by triggering the T cell receptor (TcR)‐CD3 complex leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins. To date, there has been no direct evidence on the identity of the tyrosine kinase activity implicated in this signaling pathway. In this study, we demonstrate that activation of human T cells with anti‐CD3 monoclonal antibody increases tyrosine kinase activity of p56lck. This extends our previous findings which demonstrated the involvement of p56lck kinase activity in the CD2 signal transduction pathway. The results from peripheral blood lymphocytes and Jurkat cell line showed in both cases an early and transient change in the specific activity of p56lck, followed by a shift to a higher apparent molecular mass. Therefore, to test directly the role of TcR‐CD3 in CD2‐induced activation of p56lck, we utilized mutant variants of the Jurkat cell line lacking in cell surface TcR‐CD3. We found that cell surface expression of TcR‐CD3 is not required for the activation of p56lck via CD2 but is necessary for the appearance of the reduced‐electrophoretic‐mobility form of p56lck observed after CD2 triggering. By isolating CD45‐ mutants from Jurkat cells, we observed that surface expression of the tyrosine phosphatase CD45 is required in order to increase p56lck activity following CD2 stimulation, while CD4‐induced activation of the kinase remained unchanged. These data provide evidence for a specific functional linkage between CD2 and p56lck, in which CD45 may play an essential role.
Antigen presentation for T cell interleukin‐2 secretion is a late acquisition of neonatal B cells
Authors: Morris Jennifer F. , Hoyer Jeffry T. , Pierce Susan K.
Published: March 2007

The ability of B lymphocytes to process and present antigen to helper T cells is essential to initiate T cell‐B cell interactions in humoral immune responses. Here we describe the developmental acquisition of the antigen‐presenting function of B cells as measured by the ability of B cells to stimulate a T cell hybrid to interleukin (IL)‐2 secretion. Neonatal splenic B cells are not adult‐like in their ability to process and present the model protein antigen pigeon cytochrome (Pc), which enters the B cell through fluid‐phase pinocytosis, until 21 to 28 days of life. The ability of neonatal B cells to process and present antigen which enters the cell bound to surface Ig is not adult‐like until 28 days of age. When neonatal B cells acquire antigen‐presenting cell (APC) function, surface IgM facilitates antigen processing. The delayed acquisition of APC function cannot be accounted for solely by a deficiency in major histocompatibility complex MHC class II, ICAM‐1, or LFA‐1 as neonatal B cells express adult levels of these molecules by 7–14 days after birth. Moreover, the ability of neonatal B cells to present a peptide fragment of Pc which does not require processing is adult like by day 14. Furthermore, neonatal B cells are capable of binding, internalizing and degrading radiolabeled antigen, suggesting a more subtle level of regulation. In contrast to neonatal B cells, immature B cells in the adult bone marrow and adult B cells undergoing antigen‐driven differentiation to memory B cells, as defined by the loss of the J11D marker, are competent to process and present antigen resulting in T cell IL‐2 secretion. Thus, developing B cell subpopulations in the adult and in the neonate can be distinguished. Only neonatal B cells are deficient in their ability to stimulate T cells to IL‐2 production.
Use of Torpedo‐mouse hybrid acetylcholine receptors reveals immunodominance of the α subunit in myasthenia gravis antisera
Authors: Loutrari Heleni , Tzartos Socrates J. , Claudio Toni
Published: March 2007

The nicotinic acetylcholine receptor (AChR), a pentameric complex of α2βγδ subunits, is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti‐AChR antibodies are found in ∼90% of MG patients and using indirect methods (competitive binding to solubilized AChR), peptides, or synthetic peptides, the majority of these antibodies have been shown to bind to the AChR α subunit. In order to determine directly the AChR subunit specificities of MG antibodies, we employed as antigens a novel set of hybrid AChR composed of species cross‐reacting and non‐cross‐reacting subunits stably expressed in fibroblasts. Sequence similarities of homologous subunits among species can vary widely, with mammalian subunits having 87 %‐96 % identity and Torpedo‐mammalian subunits having 54 % −80 % identity. These findings are reflected in antigenic specificities, with human anti‐AChR antisera frequently recognizing mouse AChR but rarely recognizing Torpedo. By establishing separate cell lines stably expressing all‐Torpedo, all‐mouse, and different combinations of Torpedo and mouse subunits, we were able to provide the first direct evidence of a predominant anti‐α subunit specificity in MG antisera. Functional hybrid AChR stably expressed in an intact cell membrane provide us, with a system that best mimics the in vivo environment of the MG antibody in a binding assay. Such a system allows us to investigate a perplexing observation in the field: a poor correlation between the patient's clinical status and antibody titer. Those antibodies which can interfere with AChR function, such as ones with the ability to cross‐link AChR and induce their accelerated internalization and degradation (antigenic modulation) might represent a subpopulation of MG antibodies important in disease induction or maintenance. In this report, we demonstrate that wild‐type and hybrid AChR expressed in fibroblasts can be antigenically modulated by intermolecular cross‐linking antibodies as AChR are in native muscle cells. Because we can monitor dynamic interactions between AChR and MG antibodies, this system may allow us to define crucial pathogenic epitopes in MG by expressing hybrid, chimeric, and mutant AChR.
Evidence for an association between the T cell receptor/CD3 antigen complex and the CD5 antigen in human T lymphocytes
Authors: Osman Narin , Ley Steven C. , Crumpton Michael J.
Published: March 2007

In this work we report that CD5, a T cell accessory activation antigen and receptor for the B cell surface protein CD72, is associated with the T cell antigen receptor (TcR)/CD3 complex in human T lymphocytes. In vitro phosphorylation of either CD3 or CD5 immunoprecipitates prepared from CD3‐stimulated Jurkat and peripheral blood T cells in the presence of the detergent polyoxyethylene 10 oleyl ether (Brij96) showed, unexpectedly, an identical pattern of five phosphopolypeptides of 70, 59, 56, 21 and 18 kDa, respectively. Peptide mapping of the five bands demonstrated that the same protein kinase substrates co‐precipitated with both CD3 and CD5 and that the majority of the protein phosphorylation occurred on tyrosine residues. These data suggested that the TcR/CD3 complex and and the CD5 antigen might be associated in T cells. Evidence to support this hypothesis was obtained from analysis of immunoprecipitates prepared from surface‐iodinated T cells. Bands characteristic of the TcR and CD3 antigens were identified in CD5 immunoprecipitates and conversely, CD5 was identified in CD3 immunoprecipitates. Conformation that CD3 and CD5 co‐precipitated in the presence of Brij96 was obtained by Western blotting. Quantitative immunodepletion demonstrated that between 10 % –20 % of cell surface CD5 was associated with the TcR/CD3 complex in Brij96 detergent lysates of human T cells and, furthermore, that this association was independent of T cell activation. The association of these two receptors provides a possible physical basis for the accessory role of the CD5 antigen in T cell activation.
Functional and molecular characterization of single, (4‐hydroxy‐3‐nitrophenyl)acetyl (NP)‐specific, IgG1+ B cells from antibody‐secreting and memory B cell pathways in the C57BL/6 immune response to NP
Authors: Lalor Paul A. , Nossal Gustav J. V. , Sanderson Ralph D. , McHeyzer‐Williams Michael G.
Published: March 2007

We have used multiparameter flow cytometry to identify a population of IgG1+ IgM− antigen‐specific B cells which emerges in spleens of C57BL/6 mice following immunization with the hapten, (4‐hydroxy‐3‐nitrophenyl)acetyl (NP). Characterization of the specificities of IgG1 antibodies produced by single, sorted IgG1+ NP+ cells in both Elispot assays and in microcultures containing lipo‐polysaccharide, interleukin (IL)‐2, IL‐4 and IL‐5 indicates that the splenic IgG1+ NP+ B cell population includes both IgG1 anti‐NP antibody‐secreting cells and non‐secreting, IgG1+ memory B cells. Each functionally discrete population of IgG1+ B cells expresses a distinctive surface phenotype defined by a wide range of B cell markers. In particular, antibody‐secreting, IgG1+ cells were uniquely identified by co‐expression of the matrix receptor, syndecan. The NP‐specific B cell population emerging in the day 7 primary response was assessed for clonotypic diversity by amplification and direct sequencing of the rearranged V186.2 heavy chain variable region gene expressed by single, ex vivo IgG1+ NP+ λ+ B cells. Memory B cell clones, distinguished by junctional diversity, carried either no mutation or a single mutation within rearranged V186.2, suggesting isolation of these cells at the onset of the hypermutation mechanism. This novel approach, therefore, allows the direct and unambiguous identification and characterization of individual B cell clonotypes during their initial selection and activation in antibody responses in vivo.