bluuebunny/crossref_metadata_2025 huggingface dataset:
Human T cell autoimmunity against myelin basic protein: CD4+ cells recognizing epitopes of the T cell receptor β chain from a myelin basic protein‐specific T cell clone
Authors: Saruhan‐Direskeneli Güher , Weber Frank , Meinl Edgar , Pette Martin , Giegerich Gerhard , Hinkkanen Ari , Epplen Jörg T. , Hohlfeld Reinhard , Wekerle Hartmut
Published: March 2007

We have investigated whether the normal immune system contains T cells that are able to recognize T cell receptor (TcR) determinants of autologous autoantigen‐specific T cells. The T cell clone HW. BP3, specific for myelin basic protein (MBP) was isolated from a healthy donor. HW.BP3 is restricted by HLA‐DR2a, and reacts to human MBP 139‐153. The expressed α β TcR genes of HW.BP3 were cloned and sequenced, and the sequences analyzed for potential T cell epitopes. Two synthetic peptides, one from the VDJβ junctional (β1) and one from theVβ region (β2) of the TcR of IIW.BP3, were used to select four TcR peptide‐specific T cell lines from the donor of HW.BP3. All anti‐TcR lines had the phenotype CD3+/CD4+/HLA‐DR+/CD25−/CD45RO−, and recognized the antigen in the context of HLA‐DR. Three anti‐TcR lines, which had been selected for reactivity to peptide pi, recognized exclusively this peptideβ1, restricted by HLA‐DR2b. One anti‐TcR line, selected for peptide (β2, responded to both peptides pi and P2 when presented by autologous blood mononuclear cells, but not by HLA‐DR2a‐ or HLA‐DR2b‐transfected L cells.

All TcR peptide‐specific T cell lines were efficiently cytotoxic. They specifically lysed autologous macrophages or HW.BP3 line cells in the presence of exogenous peptide antigen. In contrast, HW.BP3 did not present endogenous TcR peptides to the anti‐TcR lines. The results demonstrate that the normal human immune system contains not only autoantigen‐specific T cells, but also T cells that recognize antigenic determinants of autologous autoreactive TcR.
The CD2 and CD28 adhesion molecules induce long‐term autocrine proliferation of CD4+ T cells
Authors: Costello Régis , Cerdan Chantal , Pavon Christine , Brailly Hervé , Hurpin Christian , Mawas Claude , Olive Daniel
Published: March 2007

In vitro human T lymphocyte activation requires two‐signal triggering delivered by lectins, phorbol esters or antibodies directed against surface molecules. Stimulation of adhesion molecules by CD2 and/or CD28 antibodies defines alternative activation pathways. Activation by CD2 + CD28 monoclonal antibodies induces high‐level, long‐lasting and monocyte‐independent proliferation of highly purified T cells. Limiting dilution cultures showed that CD28 in association with CD2 or CD3, without addition of exogenous cytokines, induced single‐cell proliferation. CD2 + CD28 stimulation induced long‐term interleukin (IL)‐2‐dependent autocrine proliferation of CD4+ T cell clones. We tried to elucidate this long‐term proliferation by evaluating cytokine secretion and cytokine dependency. CD28 associated to CD3 or CD2 induced high levels of IL‐2, tumor necrosis factor (TNF) and IL‐4 secretion for 10 days, in contrast to CD3 alone which induced only TNF secretion. Cytokines of the monocytic lineage were also secreted, such as colony‐stimulating factor‐1, granulocyte macrophage colony‐stimulating factor or IL‐1, the latter being more specific of CD2 + CD28 activation. Blocking antibodies confirmed the crucial role of IL‐2 in CD2 + CD28 activation. Anti‐IL‐4, anti‐IL‐7 receptor or anti‐TNF antibodies had no effect on proliferation. Stimulation with CD2 + CD28 induced long‐term autocrine (at least for IL‐2) proliferation for CD4+ T cells, with no evidence for the implication of another cytokine among those tested other than IL‐2. This represents a model for long‐term autocrine growth for non‐leukemic cells.
Isolation of an HLA‐A2.1 extracted human minor histocompatibility peptide
Authors: de Bueger Marleen , Verreck Frank , Blokland Els , Drijfhout Jan Wouter , Amons Reinout , Koning Frits , Goulmy Els
Published: March 2007

Purified HLA‐A2.1 molecules obtained by affinity chromatography of 6 × 1010 Epstein Barr virus (EBV)‐transformed B lymphocytes were used in an attempt to isolate the human HLA‐A2.1‐restricted minor histocompatibility (H) peptides H‐Y and HA‐2. Fraction 18 of the high‐performance liquid chromatography (HPLC)‐separated HLA‐A2.1 peptide pool was found to contain the natural HA‐2 peptide. An HA‐2‐specific, HLA‐A2.1‐restricted cytotoxic T lymphocyte clone lysed HLA‐A2.1+ HA‐2− EBV‐transformed B lymphocyte cell lines reproducibly and in a concentration‐dependent fashion in the presence of fraction 18, but not in the presence of other HPLC fractions. By contrast, H‐Y sensitizing activity was not found in any fraction. Amino acid sequencing of peptide fraction 18 revealed a mixture of peptides with maximal length of nine amino acids, in which the presence of Leu at positions 2 and 9 was dominant. Surprisingly, the HA‐2 peptide could not be mimicked by any of the peptide mixtures synthesized according to the amino acid sequences found in fraction 18. Our failure to obtain the actual amino acid sequence of the human minor H peptide HA‐2 from a peptide pool with the established pattern for binding to HLA‐A2.1 may indicate that this CTL defined minor H peptide does not represent an abundant HLA‐A2.1 binding peptide.
Mercuric chloride down‐regulates T cell interferon‐γ Production in Brown Norway but not in Lewis rats; role of glutathione
Authors: Van Der Meide Peter H. , De Labie Miranda C. D. C. , Botman Caroline A. D. , Van Bennekom Wout P. , Olsson Tomas , Aten Jan , Weening Jan J.
Published: March 2007

Injection of a low dose of mercuric chloride into Brown Norway (BN) rats caused a marked decrease in the concanavalin A (ConA)‐induced generation of interferon‐γ‐producing cells (IFN‐γ pc) in spleen cell cultures prepared 1 h after mercury administration. A second injection 48 h later caused a further diminution of IFN‐γ pc down to 30% of the number generated in splenocyte cultures of phosphate‐buffered saline (PBS)‐injected controls. Injection of Lewis rats with either one or two doses of HgCl2 revealed no inhibitory effect on splenic IFN‐γ production.

The presence of the reduced form of glutathione (GSH) in the culture medium was found to be essential in these experiments. In the absence of GSH there was an overall 20‐fold reduction of the number of IFN‐γ pc in splenocyte cultures of normal or PBS‐injected rats, which was further reduced to a 60‐ to 70‐fold‐lower level in cultures of rats exposed to HgCl2. This mercury‐mediated extra reduction could be fully reversed with an excess (2 mM) of GSH in Lewis but not in BN splenocyte cultures. Since the bivalent Hg2+ ion is known to bind to and inactivate sulfydryl groups of proteins and low molecular weight thiols, most notably GSH, we investigated a possible role for thiols in IFN‐γ production. It was found that the generation of IFN‐y pc in normal BN and Lewis splenocyte cultures was strongly dependent on GSH or its precursor cysteine in the culture medium. Other thiol compounds were also effective but disulfides were completely inactive. Depletion of intracellular GSH in ConA‐stimulated splenocytes by buthionine sulfoximide (BSO), an inhibitor of de novo GSH biosynthesis, strongly inhibited the generation of IFN‐γ pc. The inhibitory effect of BSO was not abolished by the addition of interleukin‐2 (IL‐2), but was mimicked with antibodies directed to the IL‐2 receptor. The data stress the importance of GSH in the enhancement of IL‐2‐mediated IFN‐γ production and are most consistent with a model in which mercury interferes with T cell IFN‐γ production by affecting the intracellular availability of GSH. The strain‐specific susceptibility to mercury‐mediated inhibition of IFN‐γ production is discussed.
Evidence for a preferential Vβ usage by the T cells which adoptively transfer diabetes in NOD mice
Authors: Edouard Philippe , Thivolet Charles , Bedossa Pierre , Olivi Martine , Legrand Béatrice , Bendelac Albert , And Jean‐François Bach , Carnaud Claude
Published: March 2007

Non‐obese diabetic (NOD) mice become spontaneously diabetic as a result of a genetically programmed autoimmune process mediated by autoreactive T lymphocytes and directed against β cell antigen(s). Studies dealing with T cell receptor (TcR) variable (V) gene usage by such autoreactive T lymphocytes have given contrasted results. Various reasons may explain these discrepancies: the multiplicity of antigenic epitopes putatively recognized by T cells, the ambiguity between specifically committed T cells and passenger lymphocytes homing randomly to the pancreas, the necessarily limited size of the T cell clone panels which have been analyzed for TcR rearrangements and, last but not least, the flexibility of T cell repertoires. To circumvent some of these difficulties, we have decided to concentrate upon the T cell population present in diseased animals and capable of transferring diabetes into young naive NOD recipients. This population, composed of CD4+ and CD8+ T cells, is presumably committed against the relevant β cell antigens and is the most likely to reveal a bias in V gene usage if such a bias does indeed exist.

To find out whether certain Vβ genes are more frequently used than others by such pathogenic T cells, T lymphocytes from diabetic donors have been depleted in vitro of defined Vβ subsets before being reinoculated into permissive recipients. Out of four Vβ families probed under such conditions, three (Vβ8, Vβ5 and Vβ11 are neutral. Their absence neither increases nor reduces the final incidence of successful transfers, indicating that these gene segments are not preferentially used. In contrast, the depletion of Vβ6‐positiveT cells results in a severe reduction of transfers, suggesting that Vβ6 gene is used with a relatively high frequency by diabetogenic CD4+ and/or CD8+ T cells. To define more precisely which subset uses Vβ6 gene preferentially, we have performed mixing experiments with deleted and intact subsets. The results, based on disease transfer and insulitis severity, indicate that the Vβ6 bias affects predominantly the CD4+ subset. Thus, at variance with several studies concluding that V gene usage in NOD mice is heterogeneous, our present data suggest that disease transferring T cells use a relatively restricted set of Vβ genes.
T cell clones specific for p21 ras‐derived peptides: Characterization of their fine specificity and HLA restriction
Authors: Gedde‐Dahl Tobias , Fossum Beate , Eriksen Jon Amund , Thorsby Erik , Gaudernack Gustav
Published: March 2007

Peptides corresponding to the mutated regions of the oneoprotein p21 ras are immunogenic and capable of eliciting HLA class II‐restricted T cell responses. Here we report studies on the fine specificity of four T lymphocyte clones (TLC) from a single donor, using various truncated peptides derived from the residues 6–19 of p21 ras and a panel of well‐characterized HLA homozygous cells as antigen‐presenting cells. Putative minimum peptides of nine or ten amino acids could be defined for each TLC. Two of the TLC recognized peptides presented by DR2, and the two others recognized peptides presented by DQ6. Some notable differences in the requirement for certain amino acids were seen between the DR‐and DQ‐restricted TLC. Thus, Ser at residue 17 was required for stimulation of the DQ6‐but not the DR2‐restricted TLC. Val at residue 8 was essential for stimulation of all TLC, whereas one of the DR2‐restricted TLC also required Val at residue 7. Some peptides which were nonstimulatory were still capable of binding to DQ6 molecules in peptide competition experiments. The results may be of importance for potential immunotherapy of cancer where transforming ras oncoproteins are involved.
RANTES and related chemokines activate human basophil granulocytes through different G protein‐coupled receptors
Authors: Bischoff Stephan C. , Krieger Martin , Brunner Thomas , Rot Antal , Tscharner Vincenz V. , Marco Baggiolini  , Dahinden Clemens A.
Published: March 2007

Chemotactic cytokines related to interleukin‐8 (IL‐8; CXC‐chemokines) or monocyte chemotactic protein‐1 (MCP‐1; CC‐chemokines) have been shown to stimulate human basophils, and are considered important tissue‐derived mediators of inflammation. We have studied the effects of four CC‐chemokines and show that MCP‐1, RANTES (regulated on activation, normal T expressed and secreted) and macrophage inflammatory protein‐lα (MlP‐lα) are potent basophil agonists inducing a rapid change of cytosolic free calcium ([Ca2+]i), the release of histamine and sulfido‐leukotrienes, and chemotaxis. MCP‐1 was the most potent stimulus of release, and the only chemokine that induced marked exocytosis in basophils without pretreatment with interleukin‐3. RANTES was the strongest stimulus of chemotaxis, but only a moderate stimulus of release. MIP‐lα elicited relatively weak chemotaxis and release responses, but was effective at considerably lower concentrations than MCP‐1 and RANTES. MIP‐1β, by contrast, despite its high homology to MlP‐lα, was totally inactive. Normodense human eosinophils, tested for comparison, responded in a similar fashion to RANTES and MIP‐lα, but were unresponsive to MCP‐1 and MIP‐lβ. All CC‐chemokines except MIP‐lβ induced a similar rapid and transient rise of [Ca2+]| that was sensitive to pertussis toxin, indicating that they activate basophils via G‐protein‐coupled receptors. Cross‐desensensitization experiments indicate that basophils bear different CC‐chemokine receptors. Some interact selectively with MCP‐1 or RANTES, while others are shared by RANTES and MlP‐lα.
CD20 monoclonal antibodies down‐regulate IgM at the surface of B cells
Authors: Bourget Isabelle , Breittmayer Jean‐Philippe , Grenier‐Brossette Nicole , Cousin Jean‐Louis
Published: March 2007

The CD20 molecule is a phosphoprotein expressed on the surface of B lymphocytes that plays a role in the regulation of B cell proliferation and differentiation. In this study it was found that monoclonal antibodies (mAb) directed to CD20 decrease the expression of IgM at the surface of normal human B lymphocytes and B cell lines. This effect was time‐dependent with a half‐time of about 5 h. Incubation of B cells with CD20 mAb B1 did not affect the steady‐state level of IgM mRNA, suggesting that it acts at a nontranscriptional stage. Phorbol esters also produced inhibitory effect on surface IgM expression. Staurosporine reversed both the phorbol ester‐ and the CD20‐induced down‐regulation. Genistein did not reversed the down‐regulation induced by the CD20 mAb Bl. CD20 most likely triggers a protein kinase C‐dependent pathway to down‐regulate slgM. CD20 mAb also counteracted the interleukin‐4 (IL‐4)‐induced up‐regulation of slgM. The ability of anti‐IgM to mobilize intracellular calcium was reduced in slgM down‐regulated cells, suggesting that B cells activation through the antigen receptor may be negatively regulated by CD20 and positively by IL‐4.
Antigen‐specific therapy of experimental myasthenia gravis with acetylcholine receptor‐gelonin conjugates in vivo
Authors: Urbatsch Ina L. , Sterz Raimund K. M. , Peper Klaus , Trommer Wolfgang E.
Published: March 2007

Rats suffering from experimental autoimmune myasthenia gravis (EAMG) induced by previous immunization with foreign acetylcholine receptor (AChR) were treated with AChR‐toxin conjugates using the plant toxin gelonin. This led to a marked improvement of clinical symptoms as well as a significant increase in functional AChR compared to untreated rats with EAMG as determined 6 to 10 weeks later. No therapeutic effect was observed after treatment with gelonin or AChR alone. The immune response to irrelevant control antigens was not altered by this treatment.
T and B cell immune response to a 55‐kDa endothelial cell‐derived antigen in severe asthma
Authors: Lassalle Philippe , Delneste Yves , Gosset Philippe , Gras‐Masse HÉLÈNe , Wallaert Benoit , Tonnel André‐Bernard
Published: March 2007

Current concepts on the pathogenesis of chronic asthma emphasize the role of several inflammatory cell populations and their respective mediators that interact in a complex network. However, beside inflammatory cells, lymphocytes are also present in asthmatic airways. Although little is known about their involvement in asthma, it has been suggested that lymphocytes may participate in the development of chronic inflammation either through lymphokine secretion or through antibody production. In this study, we describe circulating IgG autoantibodies, directed against a common 55‐kDa antigen shared by platelets and cultured endothelial cells, and found in 34 out of 97 asthmatic patients. Among epidemiological, clinical and biological characteristics of these asthmatic patients, the anti‐55‐kDa antigen antibodies are mainly restricted to patients with negative cutaneous prick tests (p = 0.0014), and corticosteroid‐dependent asthma (p = 0.0036). These antibodies were also detected in a few patients with autoimmune disorders like systemic lupus erythematosus (3/30) or rheumatoid arthritis (2/36). Both platelet and endothelial cell antigens were cross‐reactive, had an isoelectric point between 8.0 and 9.0, were unsensitive to reducing agents such as 2‐mercaptoethanol. and were not present on either platelet or endothelial cell surface, as determined by immunostaining assay. [3H] Thymidine incorporation assay with peripheral blood mononuclear cells from patients in the presence or in the absence of 55‐kDa antigen, purified from nitrocellulose sheets demonstrated a specific incorporation in 6 out of 13 patients with circulating anti‐55‐kDa antigen antibodies, with index values ranging from 12 to 3. Such a T cell reactivity has also been observed in 3 out to 17 patients without detectable serum anti‐55‐kDa antigen antibodies. Moreover, a significant correlation was found between index values of antigen‐specific T cell reactivity and the forced expiratory volume in one second (r = 0.544, p = 0.003). Our data indicate that the detection of such antibodies allows to distinguish a subgroup of asthmatics in terms of severity and to suggest a relationship between clinical severity and T and B cell autoreactivity to the 55‐kDa platelet/endothelial cell antigen.
Increased and highly stable levels of functional soluble interleukin‐6 receptor in sera of patients with monoclonal gammopathy
Authors: Gaillard Jean‐Philippe , Bataille Régis , Brailly Hervé , Zuber Caroline , Yasukawa Kiyoshi , Attal Michel , Maruo Naoko , Taga Tetsuya , Kishimoto Tadamitsu , Klein Bernard
Published: March 2007

Soluble human interleukin‐6 receptor (sIL‐6R) was measured in the serum of 30 healthy individuals, 32 individuals with monoclonal gammopathy of undetermined significance (MGUS), 20 patients with early multiple myeloma (MM) and 54 patients with overt MM. The serum activity recognized by an immunoradiometric assay was determined to be sIL‐6R, because of its binding capacity to IL‐6 and its molecular mass of 55 kDa.

All sera of healthy individuals contained sIL‐6R (mean value: 89 ng/ml, range 17‐300 ng/ml). Serum sIL‐6R levels were increased by 51% in patients with MGUS (mean value: 135 ng/ml, p<0.005), by 44% in patients with early myeloma (mean value: 128 ng/ml, p<0.001) and by 116 % in patients with overt MM (mean value: 193 ng/ml, p<0.001). In patients with MM, a complete lack of correlation (p>0.7) was found between serum sIL‐6R levels and other previously recognized prognostic factors in this disease, particularly serum IL‐6 levels and those factors related to tumor cell mass. The independence of serum sIL‐6R levels on tumor cell mass was directly demonstrated by studying four patients with MM treated with autologous bone marrow transplantation for periods of between 320 and 760 days. These levels were found to be remarkably stable and constant, independent of whether patients relapsed or achieved complete remission. Finally, physiological concentrations of sIL‐6R were found to increase by tenfold the sensitivity of human myeloma cell lines to IL‐6. These observations suggest a high control of the sIL‐6R level in vivo, and, possibly, an important functional role of this circulating protein in patients with monoclonal gammopathies.
Tumor necrosis factor induces enhanced responses to platelet‐activating factor and differentiation in human monocytic Mono Mac 6 cells
Authors: Webera Christian , Aepfelbacher Martin , Haag Heinrich , Ziegler‐Heitbrock H. W. Liims , Weber Peter C.
Published: March 2007

Human Mono Mac 6 cells exhibit characterstics of mature blood monocytes. Treatment of these cells with human recombinant human tumor necrosis factor (TNF)resulted in an increase in phagocytosis and phorbol myristate acetate stimulated superoxide anion production at 12 h and growth retardation occurring at 24 h. Moreover, TNF induced a moderate increase of CD14 surface antigen expression, used as a phenotypic marker of monocyte/macrophage differentiation. Platelet‐activating factor (PAF) stimulated a rapid rise in cytosolic free Ca++ ([Ca++]j) of 308 & 93 nM inTNF‐treated cells compared to untreated cells (33 ± 8 nM, n = 4). The effect of TNF was dose and time dependent, evident after 12 h and maximal at 48 h. The enhanced PAF‐induced [Ca++]i rise was inhibited by the PAF receptor antagonist L‐659,989 and EGTA, indicating receptor‐dependent Ca++ influx. Furthermore, L‐659,989 and PAF inhibited specific 3H‐labeled PAF binding inTNF‐treated, but not in untreated cells. Consistently, PAF stimulated arachidonic acid release only in TNF‐treated cells. Preincubation of cells with anti‐TNF monoclonal antibodies abolished TNF‐induced effects, but failed to block lipopolysaccharide (LPS) effects. Distinct mechanisms of action by LPS were reflected by the different ability to induce surface antigen expression. In conclusion, the enhancement of PAF responses by TNF, associated with functional characteristics of differentiation in Mono Mac 6 cells, may represent a specific mechanism of cooperative interaction between PAF and TNF in inflammation, sepsis, immunoregulation and atherogenesis.
Distinct patterns of early response gene expression and proliferation in mouse mast cells stimulated by stem cell factor, interleukin‐3, or IgE and antigen
Authors: Tsai Mindy , Tam See‐Ying , Galli Stephen J.
Published: March 2007

Stem cell factor (SCF) is encoded at the Sl locus of the mouse and is the ligand for the c‐kit receptor. Recombinant rat SCF164 (rrSCF164) induces proliferation and promotes maturation of mouse mast cells in vitro and in vivo and can also induce c‐kit receptor‐dependent mouse mast cell degranulation. We now report that in both quiescent and non‐quiescent mouse bone marrow‐derived cultured mast cells (BMCMC) rrSCF164 induces increased mRNA levels for the “early response genes” c‐fos, c‐jun and junB but has only slight effects on the expression of junD.

Recombinant mouse interleukin‐3 (IL‐3) also promotes proliferation of both quiescent and non‐quiescent BMCMC. However, IL‐3 induces increased expression of c‐fos and juriB only in quiescent BMCMC. Cross‐linking of Fee receptor type I (FcϵRI) on BMCMC by IgE and specific antigen induces a pattern of early gene expression very similar to that induced by rrSCF164. However, BMCMC stimulated through the FcϵRI did not proliferate and, in comparison to control BMCMC, exhibited significantly decreased proliferation in response to rrSCF164 or IL‐3. These results indicate that stimulation of BMCMC proliferation by IL‐3 or rrSCF164 induces distinct patterns of early response gene expression and suggest that the proliferative effects of these growth factors may be mediated through distinct signal transduction pathways. Our data also point to previously unappreciated similarities between the effects of signaling through the c‐kit receptor or the FcϵRI on mast cell expression of fos and jun genes.
In vivo induction of the nitric oxide pathway in hepatocytes after injection with irradiated malaria sporozoites, malaria blood parasites or adjuvants
Authors: Nussler Andreas K. , Rénia Laurent , Pasquetto Valerie , Miltgen Francois , Matile Hughes , Mazier Dominique
Published: March 2007

The mechanisms responsible for malarial immunity induced by repetitive injections of X‐irradiated sporozoites have not been fully established. We demonstrate here that a single injection of irradiated sporozoites induced, as soon as 24 h after, a non‐permissive state to hepatocyte reinfection with sporozoites in vitro. The same effect was observed when malarial blood forms, irradiated promastigotes of Leishmania infantum, adjuvants (muramyl dipeptide, poly acidylic uridylic) or interferon‐γ was injected. Activation of the nitric oxide (NO) pathway in the hepatocyte by these factors was found to be responsible for hepatocyte refractory status. Additionally, this metabolic pathway is involved in protection given by repeated injections of irradiated sporozoites since protection could be reversed by treating mice at the time of sporozoite challenge with a competitive inhibitor (NG‐monomethyl‐L‐arginine) of the NO pathway. These results suggest that, in view of an antisporozoite vaccine, further studies are needed to find out how to activate specifically a long‐lasting nonspecific immune response.
Streptavidin blocks immune reactions mediated by fibronectin‐VLA‐5 recognition through an Arg‐Gly‐Asp mimicking site
Authors: Alon Ronen , Hershkoviz Rami , Bayer Edward A. , Wilchek Meir , Lider Ofer
Published: March 2007

Streptavidin is a biotin‐binding analogue of egg‐white avidin which is secreted by the bacterium Streptomyces avidinii. We have recently reported that streptavidin contains an Arg‐Tyr‐Asp‐Ser (RYDS) sequence which exhibits structural homology to the Arg‐Gly‐Asp‐Ser (RGDS) cell adhesion domain of fibronectin and other matrix‐associated glycoproteins. Competition studies with RGD peptides indicated that streptavidin binds to cells via this site and that the binding is independent of biotin recognition. Since the RGD‐containing peptide has been shown to play a key role in integrin‐mediated cell adhesion, we assumed that streptavidin may utilize the RYDS site to bind to immune cells and thereby abrogate their adhesion‐dependent functions. We now report that streptavidin modulates several atrix‐dependent interactions of immune cells. In this context, immobilized streptavidin was found to support activated human CD4+ T cell adhesion in an RGD‐specific, α5β1 dependent manner. In addition, soluble streptavidin (the commercially available or biotin‐blocked forms) inhibited T cell adhesion to fibronectin and interfered with its co‐stimulatory effect on tumor necrosis factor‐a secretion by co‐cultures of CD4+ T cells and macrophages. These results suggest that streptavidin is a novel example of a bacterial protein which utilizes RGD mimicry to interfere with integrin‐mediated immune responses.
In situ analyses of in ovo graft‐vs.‐host reaction induced by thymic nurse cell lymphocytes
Authors: Rieker Theresa , Penninger Josef , Hala Karel , Cooper Max D. , Wick Georg
Published: March 2007

In both mammalian and avian systems, thymic nurse cells (TNC) have been shown to harbor a heterogeneous population of T lymphocytes (TNC‐L) some of which exhibit a postselectional phenotype. By tranplanting micromanipulated single chicken TNC onto the chorionallantoic membrane (CAM) of major histocompatibility complex (MHC)‐disparate embryos, an experimental system which allows for the detection of lymphocytes with graft‐vs.‐host (GVH) reactivity, we demonstrate here that TNC enclose lymphocytes that can develop into both CD4+ single‐positive (sp) and CD8+ sp, T cell receptor (TcR)αβ+, or TcRγδ+ cells. This finding was additionally confirmed by serial transfer of primary expanded alloreactive T cells onto the CAM of secondary hosts. All donor TNC‐L expressed MHC class II molecules and the interleukin‐2 receptor a chain in primary and secondary GVH reactions. Furthermore, we observed selective accumulation of CD8+ and TcRγδ+ host lymphocytes in the CAM upon the induction of a local GVH reaction, most probably as a consequence of the pathological alteration of the epithelium.
A subset of anti‐CD21 antibodies promote the rescue of germinal center B cells from apoptosis
Authors: Bonnefoy Jean‐Yves , Henchoz Sybille , Hardie Deborah , Holder Michelle J. , Gordon John
Published: March 2007

Germinal center cells (GCC) are programmed to die by apoptosis unless they receive a positive signal for rescue. The primary signal in vivo is believed to be dependent on interaction with antigen held as immune complexes on follicular dendritic cells (FDC), a subset of which express large amounts of CD23, a low‐affinity receptor for IgE. Recombinant soluble CD23 (sCD23) and interleu‐kin‐1 have been found to potentiate the survival of GCC in vitro. Recently, CD23 was shown to interact specifically with a ligand other than IgE, namely CD21 (CR2/Epstein‐Barr virus receptor). In the present study, we show that a subset of anti‐CD21 monoclonal antibodies behave similarly to soluble CD23 in their effect on GCC inasmuch as they: (i) diminish the occurrence of apoptosis; (ii) promote a plasmacytoid appearance in rescued cells; (in) up‐regulate expression of the Bcl‐2 proto‐oncogene. These findings indicate that FDC‐derived CD23 exerts its effect on GCC via CD21.
Interleukin 6 is not required for antigen‐specific antibody responses by human B cells
Authors: Costelloe Katherine E. , Smith Susan H. , Callard Robin E.
Published: March 2007

Interleukin‐6 (IL‐6) is a late‐acting differentiation factor for human B cells activated by polyclonal mitogens such as pokeweed mitogen (PWM) and Staphylococcus aureus Cowan strain I, but its role in specific antibody responses has not been established. We show here that IL‐6 has no consistent effect on specific antibody responses by tonsillar mononuclear cells (TMC) stimulated with influenza virus. A blocking IL‐6 antibody also had no effect on antibody production, suggesting that endogenous IL‐6 production was not required. In control experiments, this antibody inhibited PWM‐stimulated immunoglobulin secretion and proliferation of the IL‐6‐dependent B cell line B9. A requirement for IL‐6 in responses of unfractionated TMC may have been disguised by the presence of Tcells. To overcome this problem, we investigated the effect of IL‐6 on specific antibody production by T‐depleted B cells stimulated with antigen in the presence of IL‐2, which is a Tcell replacing factor (TRF) for human B cells. Specific antibody production was restored by IL‐2, but not IL‐6. Neither IL‐6 nor anti‐IL‐6 antibody had any consistent effect on specific antibody production by purified B cells stimulated with antigen and TRF. These experiments show that IL‐6 does not have a significant role in antigen (influenza virus)‐specific antibody responses by human B lymphocytes.
Eosinophils infiltrating interleukin‐5 gene‐transfected tumors do not suppress tumor growth
Authors: Krüger‐Krasagakes Sabine , Li Weiqun , Richter GÜNther , Diamantstein Tibor , Blankenstein Thomas
Published: March 2007

Tumor‐associated eosinophils have been observed in human tumors and in experimental tumor models, but their function is poorly understood. To study the role of eosinophils during tumor growth, the plasmacytoma J558L and the mammary adenocarcinoma TS/A were transfected with an expression vector encoding the murine gene for interleukin‐5 (IL‐5), a cytokine inducing proliferation and activation of eosinophils. Injection of parental cells, mock‐transfectants and IL‐5‐producing cells into syngeneic mice showed that local IL‐5 secretion induced rapid tumor infiltration by eosinophils, as evidenced by immunohistochemical staining, but nevertheless did not alter the tumor growth kinetics of IL‐5 transfectants. Therefore, the mere presence of IL‐5 and eosinophils was not sufficient to induce a protective host immune response.
Interleukin‐4 and interleukin‐10 inhibit nitric oxide‐dependent macrophage killing of Candida albicans
Authors: Cenci Elio , Romani Luigina , Mencacci Antonella , Spaccapelo Roberta , Schiaffella Enrico , Puccetti Paolo , Bistoni Francesco
Published: March 2007

Mouse peritoneal and splenic macrophages treated with interferon‐γ (IFN‐γ) and infected with the yeast Candida albicans expressed high fungicidal activity in vitro that correlated with increased nitrite concentrations in culture supernatants. Both effects were reduced by an inhibitor of nitric oxide (NO) synthesis which, in vivo, impaired the animals' ability to mount a footpad reaction and clear the fungus from infected organs. Because T helper type‐2 (Th2) cytokines in candidiasis are known to limit the expression of protective Th1 functions, we tested the effect of interleukin (IL)‐4 and IL‐10 on candidacidal activity and NO production of IFN‐γ‐activated macrophages. Fungal killing and NO secretion were inhibited, in a dose‐dependent manner, by the two cytokines either separately or in combination. Impaired candidacidal activity was also demonstrable in the presence of monoiodoacetic acid, an inhibitor of phagocytosis. These data demonstrate that NO is involved in macrophage killing of C. albicans and support the notion that regulation of Th1 effector function by IL‐4 and IL‐10 might involve modulation of NO synthesis.
Regulation of CD69 expression on human natural killer cells: differential involvement of protein kinase C and protein tyrosine kinases
Authors: Borrego Francisco , Peña José , Solana Rafael
Published: March 2007

Human peripheral blood natural killer (NK) cells (CD56+, CD16+, CD3ε− lymphocytes) express CD69 after their stimulation by interleukin‐2 (IL‐2) or interferon‐α (IFN‐α). This activation antigen represents a triggering surface molecule in NK cell clones as its stimulation triggers the cytolytic machinery of these cells. However, the mechanisms regulating the expression of CD69 in NK cells are unknown despite the functional relevance of CD69 in NK cell activation. Thus, we have analyzed the role of protein kinase C (PKC) and protein tyrosine kinases (PTK) in the expression of CD69 on purified NK cells activated by IL‐2, IFN‐α, anti FcγRIII (CD16) monoclonal antibodies or by K562 target cells. We found that CD69 is induced on NK cells not only by IL‐2 and IFN‐α but also by activation of the CD 16 pathway, the interaction with NK target cells and the direct activation of PKC by phorbol 12‐myristate 13‐acetate (PMA), indicating that CD69 induction is associated to different NK activation pathways. The treatment with the PKC inhibitor staurosporine abolished the induction of CD69 induced by PMA or K562. However, it did not significantly affect CD69 induction by IL‐2, IFN‐α or CD 16 cross‐linking. This demonstrates that whereas PKC can play a central role in the regulation of CD69 expression in some instances (response to K562 cells or PMA), it does not participate in others (response to IL‐2, IFN‐α or anti CD16 monoclonal antibodies). On the other hand genistein, a competitive inhibitor of PTK enzymes, blocked the expression of CD69 induced by activation of NK cells via IL‐2 or IFN‐α receptors, CD16 and K562 receptor(s), indicating that stimulation of PTK is a common step in the signal transduction events leading to the induction of CD69 antigens after the activation of NK cells via these receptors.
Interferon‐γ and interleukin‐4 reciprocally regulate the production of monocytes/macrophages and neutrophils through a direct effect on committed monopotential bone marrow progenitor cells
Authors: Snoeck Hans‐W. , Lardon Filip , Lenjou Marc , Nys Griet , Van Bockstaele Dirk R. , Peetermans Marc E.
Published: March 2007

We studied the direct effects of interferon‐γ (IFN‐γ) in single cell colony assays of CD34+HLA‐DR++ bone marrow progenitor cells stimulated by either granu‐locyte‐colony‐stimulating factor (G‐CSF), interleukin(IL)‐3, granulocyte/macro‐phage‐colony‐stimulating factor (GM‐CSF), combinations of these CSF or medium conditioned by the 5637 human bladder carcinoma cell line. In this culture system IFN‐γ stimulated monocytic colonies (CFU‐M) no matter which CSF or CSF combination was used to support them and inhibited granulocytic colonies (CFU‐G) if they were generated in the presence of G‐CSF. IL‐4 antagonized the myelopoietic effects of IFN‐γ: the IFN‐γ induced suppression of G‐CSF‐supported CFU‐G, as well as the stimulation of CFU‐M, were reversed by IL‐4. In all cultures, IFN‐γ had a limited, but statistically non‐significant, inhibitory effect on CFU‐GM, which was not affected by the presence of IL‐4. These data show that IFN‐γ and IL‐4 reciprocally regulate the generation of myeloid cells involved in humoral (neutrophils) and cellular (macrophages) immune responses through a direct effect on monopotential myeloid progenitor cells.
Identification and analysis of cytochrome P450IID6 antigenic sites recognized by anti‐liver‐kidney microsome type‐1 antibodies (LKM1)
Authors: Yamamoto Ana M. , Cresteil Danièle , Boniface Odile , Clerc François F. , Alvarez Fernando
Published: March 2007

Anti‐liver‐kidney microsome type‐1 antibodies (LKM1), present in sera from a group of patients with autoimmune hepatitis, are directed against P450IID6. Previous work, using cDNA constructions spanning most of the P450IID6 protein defined the main immunogenic site between the amino acids (aa), 254–271 and predicted the presence of other putative immunogenic sites in the molecule. Fusion proteins from new cDNA constructions, spanning so‐far‐untested regions between aa 1–125 and 431–522, were not recognized by LKM1‐positive sera. Synthetic peptides, representing sequences from putative immunogenic regions or previously untested regions, allowed a precise definition of four antigenic sites located between peptides 257–269, 321–351, 373–389 and 410–429, which were recognized, respectively, by 14, 8, 1 and 2 out of 15 LKM1‐positive sera tested. The minimal sequence of the main antigenic site (peptide 257‐269) recognized by the autoantibody was established to be WDPAQPPRD (peptide 262–270). In addition, deletion and replacement experiments showed that aa 263 (Asp) was essential for the binding of the autoantibody to peptide 262–270. Analysis of the second most frequently recognized peptide between aa 321–351, was performed using peptides 321–339 and 340–351 in competitive inhibition studies. Complete elimination of antibody binding to peptide 321–351 obtained by absorption of both shorter peptides indicated that peptide 321–351 is a discontinuous antigenic site. LKM1‐positive sera reacting against peptide 321–351 recognized either both the shorter peptides or just one of them preferentially. Results of the present study suggest that the production of LKM1 antibodies is an antigen‐driven, poly‐or oligoclonal B cell response. The identification of antigenic sites will allow: (i) the development of specific diagnostic tests and (ii) further studies on the pathogenic value of LKM1 antibodies in autoimmune hepatitis.
An intermolecular mechanism of T cell help for the production of antibodies to the bacterial pathogen, Chlamydia trachomatis
Authors: Allen Judith E. , Stephens Richard S.
Published: March 2007

Antibodies that neutralize infectivity are directed at the antigenically variant major outer membrane protein (MOMP) of Chlamydia trachomatis. A vaccine for chlamydia will need to include T cell determinants that elicit T helper (Th) cells which provide help to MOMP‐specific B cells. A limited number of determinants on MOMP are able to elicit Th cells and sequence diversity in the MOMP molecule may alter T cell recognition of these determinants. We investigated whether two sequence invariant proteins of C. trachomatis that are both abundant and immunogenic could elicit T cell help for the production of antibody to MOMP. We found that outer membrane protein 2 (OMP2) but not outer membrane protein 3 (OMP3) was able to prime BALB/c mice for an anamnestic anti‐MOMP response following boost with the intact organism. This demonstration of an intermolecular mechanism of T cell help in a bacterial system has important implications for the development of a chlamydial vaccine as well as the design of vaccines for other antigenically variant non‐viral pathogens.
Phenotypically diverse mouse thymic stromal cell lines which induce proliferation and differentiation of hematopoietic cells
Authors: Faas Susan J. , Rothstein Jay L. , Kreider Brent L. , Rovera Giovanni , Knowles Barbara B.
Published: March 2007

The heterogeneity of the thymic stroma has made careful characterization of particular thymic stromal cell types difficult. To this end, we have derived a panel of cloned thymic stromal cell lines from simian virus 40 T antigen (SV40‐T antigen) transgenic mice. Based on their analysis with monoclonal antibodies that distinguish among subsets of thymic stroma cells, and on the morphology and ultrastructural features of the different clones, we suggest that our panel includes representatives of the thymic subcapsular cortex or thymic nurse cells (427.1), the deep cortex or cortical reticular cells (1308.1) and the medulla including medullary interdigitating (IDC)‐like cells (6.1.1) and medullary epithelial cells (6.1.7). A fifth cell type of undesignated but apparent medullary origin (6.1.11) was also isolated. All of the cell lines constitutively express the SV40 T antigen transgene and the class I antigens of the major histocompatibility complex (MHC), and they can be induced to express MHC class II antigens upon stimulation with recombinant interferon‐γ (IFN‐γ). These cell lines elaborate a factor(s) that induces the proliferation of cells from the fetal liver and bone marrow, but not from the neonatal thymus. A factor(s) elaborated by the 1308.1 cell line also induces the proliferation of fetal thymocytes in the absence of mitogens, phorbol esters or calcium ionophore which is augmented with the addition of recombinant interleukin‐2 (IL‐2). Analysis by reverse transcription polymerase chain reaction with primers for some mouse cytokines reveals that each of these cell lines contain granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) transcripts and that 1308.1,6.1.1 and 6.1.7 produce IL‐6 mRNA. Cell lines 1308.1 and 6.1.1 also produce IL‐7; 6.1.1 produces IL‐lβ and tumor necrosis factor (TNF)‐α while the 427.1 cell line produces IL‐5 and IFN‐γ mRNA. None of the cell lines tested express the IL‐2 receptor, IL‐2, IL‐3, IL‐4, TNF‐β or macrophage inflammatory proteins mRNA. Conditioned medium (CM) from 1308.1 and 6.1.11 induced differentiation of cells purified from the mouse fetal liver into granulocytes; 1308.1 CM also induced differentiation of the mouse hematopoietic stem cell line 32DC13(G) suggesting that the CM contains granulocyte (G)‐CSF activity. Each cell line produces GM‐CSF but the greatest activity is associated with 1308.1 and 6.1.11 CM. The availability of these well‐characterized, functional, cloned thymic stromal cells will allow a more detailed analysis of the role of each cell type in both myeloid and Tcell development.
Predominant and stable T cell responses to regions of myelin basic protein can be detected in individual patients with multiple sclerosis
Authors: Salvetti Marco , Ristori Giovanni , D'Amato Mauro , Buttinelli Carla , Falcone Marika , Fieschi Cesare , Wekerle Hartmut , Pozzilli Carlo
Published: March 2007

T lymphocytes from patients with multiple sclerosis (MS) recognize multiple myelin basic protein (MBP) epitopes. This situation complicates the design of specific immunotherapies. We investigated to which extent the T cell response to MBP is heterogeneous in single subjects in terms of preferentially recognized regions of the molecule, major histocompatibility complex (MHC) restriction, and stability over time. From each of nine patients with MS, a minimum of six MBP‐specific T lymphocyte lines (TLL) were assayed for the proliferative response to a panel of overlapping peptides, encompassing the whole MBP. Predominant Tcell recognitions of distinct MBP regions were present in three patients, all HLA‐DR2+, independently of the clinical features of their disease. Tcell reactivity was preferentially directed to residues 16‐38 in one patient. In this case the response was also stable over time, during different phases of the disease. Predominant reactivity to residues 86‐99 was detected in the two other DR2+ patients. In each of the patients with other HLA‐DR haplotypes (DR2−), as well as in three DR2+ non‐MS donors, the Tcell response to MBP appeared to be considerably more heterogeneous. The HLA restriction element varied among TLL recognizing the same MBP region, even when raised from the same individual. The genomic HLA typing, performed on the DRB1 and DRB5 genes in the DR2+ subjects, showed no obvious correspondence between preferential responses to regions of MBP and HLA‐DR2 subtypes. In this context, a simple, new method for the genomic typing of the HLA‐DRB1 gene in individuals with the HLA‐DR2 serological specificity is also described.

We conclude that predominant and stable T cell responses to a single MBP region can be detected in some patients with MS. In these individuals, the MHC restriction of the T cell recognition of predominant regions appears to be variable. Polymorphisms of the HLA‐DR2 gene products alone do not account for the selection of the dominant MBP Tcell epitope.
Genetic polymorphism of human interleukin‐1α
Authors: Bailly Sabine , Di Giovine Francesco S. , Blakemore Alexandra I. F. , Duff Gordon W.
Published: March 2007

Interleukin‐1α (IL‐1α) has been implicated in the pathogenesis of infectious, autoimmune and inflammatory diseases. There is, however, very little information on the cis‐acting sequences involved in IL‐1α regulation or whether there is any variation in the structure of the gene. It is known that intron 6 of IL‐1α shows a 5 × 46 bp tandem repeat in the genomic sequence. We have studied this region of the gene. Amplification by polymerase chain reaction showed different sized products from different individuals, most being of higher molecular weight than the expected size of 620 bp. Sequencing demonstrated that the polymorphism was due to a variable number of repeats of the 46 bp sequence. This was confirmed by restriction fragment length analysis of genomic DNA. Altogether, 72 unrelated individuals were tested and 6 alleles ranging from 5 to 18 repeats were found, the most frequent allele (62%) containing 9 repeats. This polymorphism may be of interest in gene function, since each repeat contains three potential binding sites for transcriptional factors: an SP1 site, a viral enhancer element and a glucocorticoid‐responsive element. The latter, at least, demonstrates site‐specific protein binding by electromobility shift assay. The functional significance of the polymorphism and its allelic frequency in inflammatory and autoimmune diseases are currently under investigation.
Killing of immature CD4+CD8+ thymocytes in vivo by anti‐CD3 or 5′‐(N‐ethyl)‐carboxamido‐adenosine is blocked by glucocorticoid receptor antagonist RU‐486
Authors: Jondal Mikael , Okret Sam , McConkey David
Published: March 2007

Negative selection in thymus occurs by apoptosis in CD4+CD81 cells. These immature thymocytes are readily killed, both in vitro and in vivo, by glucocorticoid treatment. Increased levels of intracellular cAMP in vitro also induce apoptosis of thymocytes and T cell receptor (TcR) stimulation potentiate cAMP responses through receptors linked to adenylic cyclase. Presently, we have tested the possibility that TcR‐mediated apoptosis in vivo may require the glucocorticoid receptor (GR) as a downstream, intracellular mediator. Use of the GR antagonist RU‐486, 24 h before and simultaneous with, anti‐CD3 or 5′‐(N‐ethyl)‐carboxamide‐adenosine (NECA) treatment, resulted in a selective inhibition of CD4+CD8+ thymocyte death. In addition, a low dose of glucocorticoid potentiated thymocyte death induced by anti‐CD3 monoclonal antibodies. These data support a model in which thymic negative selection depends on a defined set of transduction signals which potentiate the GR to become responsive to endogenous levels of glucocorticoid.