bluuebunny/crossref_metadata_2025 huggingface dataset:
A tyrosinase nonapeptide presented by HLA‐B44 is recognized on a human melanoma by autologous cytolytic T lymphocytes
Authors: Brichard Vincent G. , Herman Jean , Van Pel Aline , Wildmann Claude , Gaugler Béatrice , Wölfel Thomas , Boon Thierry , Lethé Bernard
Published: March 2007

The human tyrosinase gene has been reported previously to code for two distinct antigens recognized on HLA‐A2 melanoma cells by autologous cytolytic T lymphocytes (CTL). By stimulating lymphocytes of melanoma patient MZ2 with a subclone of the tumor cell line of this patient, we obtained a CTL clone that lysed this subclone but did not lyse other subclones of the same melanoma cell line. The sensitive melanoma subclone was found to express a much higher level of tyrosinase than the others, suggesting that the antigen recognized by the CTL might be encoded by tyrosinase. Transfection of a tyrosinase cDNA demonstrated that the CTL clone indeed recognized a tyrosinase product presented by HLA‐B*4403. The relevant antigenic peptide corresponds to residues 192–200 of the tyrosinase protein. Lymphoblastoid cells of the B*4402 subtype were not recognized by the CTL following incubation with the peptide. Nevertheless, by stimulating in vitro lymphocytes of a healthy HLA‐B*4402 donor with autologous adherent cells pulsed with the same peptide, we obtained a CTL clone which recognized tumor cells expressing tyrosinase and HLA‐B*4402. As HLA‐B44 is expressed in 24% of Caucasians, the tyrosinase‐B44 antigen may constitute a useful target for specific immunotherapy of melanoma.
Prevention of murine lupus by an I‐E α chain transgene: Protective role of I‐E α chain‐derived peptides with a high affinity to I‐Ab molecules
Authors: Iwamoto Masahiro , Ibnou‐Zekri Nabila , Araki Kimi , Izui Shozo
Published: March 2007

The expression of a transgene encoding the I‐E α chain prevents a lupus‐like autoimmune syndrome in BXSB mice. However, it had not been elucidated whether the Eαd transgene‐mediated protective effect results from I‐E expression or from the generation of I‐E α chain‐derived peptides (Eα peptide) displaying high affinity for the I‐Ab molecule. To address this question, two different BXSB lines expressing the transgene at low or high levels were crossed with lupus‐prone MRL mice; this resulted in three types of (MRL × BXSB)F1 mice, differing in the expression levels of I‐E molecules and of Eα peptides presented by I‐Ab molecules. Comparative analysis of these three (MRL × BXSB)F1 mice as well as several BXSB transgenic lines showed that the Eαd transgene‐mediated protection paralleled the expression levels of Eα peptide presented by I‐Ab molecules, but not of I‐E molecules on B cells. In addition, use of transgenic and nontransgenic double bone marrow chimeras showed a selective activation of nontransgenic B cells during I‐Ab‐restricted T cell‐dependent immune responses, while both transgenic and nontransgenic B cells were comparably activated during T cell‐independent responses. These results favor a model of autoimmunity prevention based on competition for antigen presentation, in which excessive generation of Eα peptides prevents, because of their high affinity to the I‐A molecules, activation of potential autoreactive T and B cells.
Human natural killer cell inhibitory receptors bind to HLA class I molecules
Authors: Döhring Christian , Colonna Marco
Published: March 2007

To obtain direct evidence that human natural killer cell (NK) inhibitory receptors physically interact with polymorphic determinants of major histocompatibility complex class I molecules, a receptor termed NK‐associated transcript‐2 has been produced as a soluble fusion protein with the human IgG1 Fc portion. This soluble receptor binds specifically to HLA‐C alleles with serine 77 and asparagine 80 in cell adhesion assays.
Fas (CD95)/Fas ligand interactions regulate antigen‐specific, major histocompatibility complex‐restricted T/B cell proliferative responses
Authors: Ozdemirli Metin , El‐Khatib Maan , Foote Linda C. , Wang Julia K. M. , Marshak‐Rothstein Ann , Rothstein Thomas L. , Ju Shyr‐Te
Published: March 2007

The effect of Fas ligand (FasL) cytotoxicity on T/B collaboration was examined in vitro using cloned T helper 1 cells and antigen‐pulsed, activated B cells. We compared antigen‐pulsed B cells that had been activated through different membrane receptors (IgM, CD14 and CD40) for their ability to induce T cell proliferation and to respond to T cell help. We also used a Fas‐Ig fusion protein, an inhibitor of FasL‐mediated cytotoxicity, to determine the effect of FasL cytotoxicity on the T and B cell proliferative responses. The data show that the extent of both T and B cell proliferative responses correlate with the relative resistance of activated B cell populations to FasL cytotoxicity. Moreover, both T and B cell proliferation could be enhanced by Fas‐Ig. Our results demonstrate that FasL cytotoxicity is a negative regulatory mechanism for both T and B cell proliferative responses and that Fas‐Ig can be an immunopotentiating agent for both T and B cell immunity.
γδ T cells are secondary participants in acute graft‐versus‐host reactions initiated by CD4+ αβT cells
Authors: Tsuji Sachiyo , Char David , Bucy R. Pat , Simonsen Morten , Chen Chen‐Lo H. , Cooper Max D.
Published: March 2007

To examine the role of T cell subpopulations in an acute graft‐versus‐host (GVH) reaction, γδ T cells and αβ T cells expressing one of the two prototypic Vβ gene families were negatively isolated from adult blood samples and injected into allogeneic chick embryos. CD4+ αβ T cells expressing either Vβ1 or Vβ2 receptors were equally capable of inducing acute GVH reactions, consistent with the idea that αβ T cell alloreactivity is determined by CDR3 variability. By themselves, the γδ T cells were incapable of inducing GVH reactions. However, host γδ T cells were recruited into the donor αβ T cell‐initiated lesions, where they were activated and induced to proliferate. The data suggest that γβ T cells may play a secondary role in GVH reactions.
The phenotype and fate of the antibody‐forming cells of the splenic foci
Authors: Smith Kenneth G. C. , Hewitson Timothy D. , Nossal G. J. V. , Tarlinton David M.
Published: March 2007

The first product of the humoral response to antigen is low‐affinity antibody, produced by extrafollicular foci of antibody‐forming cells (AFC) in organs such as spleen and lymph node. These cells proliferate rapidly but then undergo an equally rapid decline, so that they are present in only small numbers 14 days after immunization. We have used 6‐parameter flow cytometry to isolate and examine the characteristics of (4‐hydroxy‐5‐nitrophenyl)acetyl‐specific AFC, looking in particular for those markers that might differentiate them from cells of the intrafollicular (germinal center) arm of the T‐dependent immune response. At day 7 of the primary response, most AFC were found to express surprisingly low levels of B220, high levels of syndecan, and retain significant levels of surface IgG1. We then used enzyme‐linked immunospot assays to demonstrate that the rapid decline of these cells was not likely to be due to migration to organs such as the bone marrow. Their decline could, however, be explained by apoptosis in situ, which was demonstrated immunohistologically by nick‐end labeling.
In Th2‐biased lymphatic filarial patients, responses to purified protein derivative of Mycobacterium tuberculosis remain Th1
Authors: Sartono Erliyani , Kruize Yvonne C. M. , Kurniawan Agnes , Maizels Rick M. , Yazdanbakhsh Maria
Published: March 2007

Natural infection with filarial nematode parasites results in immune responses skewed towards T helper (Th)2, while infection with mycobacteria shows many characteristics of a Th1‐dominated response. Cytokines typifying Th1, interferon (IFN)‐γ, and Th2, interleukin (IL)‐4, were measured following stimulation of peripheral blood mononuclear cells from filarial patients with Brugia malayi adult worm antigen (BmA) and purified protein derivative of Mycobacterium tuberculosis (PPD). In response to PPD, only 1 out of 81 patients produced IL‐4, and this at an amount (4.4 pg/ml) just above the detection limit, whereas 59% of patients responded to BmA by releasing IL‐4. Conversely, substantial quantities of IFN‐γ were released in response to PPD (geometric mean 37.43 U/ml) compared to low BmA‐stimulated IFN‐γ production in the same patients (geometric mean 5.02 U/ml). These results demonstrate that the strong skewing of the cytokine environment towards Th2 in filarial patients in vivo does not influence the predominance of a Th1 type immune response to PPD.
The differentiation of human memory B cells into specific antibody‐secreting cells is CD40 independent
Authors: Silvy Anne , Lagresle Chantal , Bella Chantal , Defrance Thierry
Published: March 2007

It is generally accepted that memory B cells can be defined by their ability to produce, upon antigenic challenge, somatically mutated antibody molecules characterized by an increased affinity and by the expression of a downstream heavy chain isotype. However, the inability to isolate this particular B cell compartment has precluded the study of memory B lymphocyte physiology in man. We previously reported on the identification of an IgD− B cell subset in human tonsils that we defined as CD38− B cells, whose phenotype is highly reminiscent of that of memory B lymphocytes from the splenic marginal zone of rodents. In the present study, we developed a model of the measles virus (MV)‐specific secondary antibody response in vitro to assess the presence of memory B lymphocytes in different B cell subsets isolated from human tonsils and explore the activation requirements of human memory B cells. Our findings show that the memory B cell pool resides in the CD38− B cell subpopulation and that the differentiation of MV‐activated memory B cells into antibody‐secreting cells can be achieved upon co‐stimulation with interleukin (IL)‐2 and IL‐10, but does not require engagement of CD40. Interestingly, the CD40‐mediated signal was found to synergize with Ig‐cross‐linking agents for the proliferation of memory B cells, but strongly suppressed their capacity to differentiate along the plasmacytoid pathway. Collectively, our results suggest that the CD40 signaling pathway is instrumental for the clonal expansion of the memory B cell pool, but does not operate in the later phase of the response, which allows their maturation into antibody‐secreting cells.
Expression of monocyte chemoattractant protein‐1 and interleukin‐8 receptors on subsets of T cells: correlation with transendothelial chemotactic potential
Authors: Qin Shixin , Larosa Greg , Campbell James J. , Smith‐Heath Heidi , Kassam Nasim , Shi Xiaojie , Zeng Ling , Butcher Eugene C. , MacKay Charles R.
Published: March 2007

The differential expression of chemokine receptors may be an important mechanism for the regulation of T cell migration. To test this, we examined the expression and function of the monocyte chemoattractant protein (MCP)‐1 and interleukin (IL)‐8 receptors on various populations of T cells. Using a simple and reliable transendothelial chemotaxis assay, both MCP‐1 and IL‐8 were shown to be chemotactic for subsets of blood T cells, although the relative response varied from donor to donor. To examine receptor expression and correlate it with chemotaxis of T cell subsets, monoclonal antibodies (mAb) to the receptors were produced by immunizing mice either with synthetic peptides (MCP‐1 receptor), or with receptor transfectants (IL‐8 receptors A and B). A flow cytometric analysis of blood T cells with an anti‐MCP‐1 receptor mAb revealed low expression on the CD26hi subset and undetectable expression on other T cells. Staining of T cells with anti‐IL‐8RA and anti‐IL‐8RB showed much higher levels of expression, but only on a subset of CD3+ cells which were CD8+ and CD56−. That IL‐8 and MCP‐1 attracted distinct subsets of T cells was best illustrated using the CD26 marker, since IL‐8R+ T cells were CD26−, whereas T cells expressing detectable MCP‐1R or which responded to MCP‐1 in chemotaxis assays were CD26hi. T cells activated in vitro with anti‐CD3 up‐regulated expression of the MCP‐1 receptor, but not the IL‐8 receptors, and were attracted to MCP‐1 much more efficiently than resting T cells. These results show that there is a clear distinction between the IL‐8‐ and MCP‐1‐responsive T cell populations and that chemokine receptor expression on T cells may be regulated with respect to lineage as well as cellular activation.
Natural killer clones recognize specific soluble HLA class I molecules
Authors: Carbone Ennio , Terrazzano Giuseppe , Colonna Marco , Tuosto Loretta , Piccolella Enza , Franksson Lars , Palazzolo Giovanni , Pérez‐Villar Juan José , Fontana Silvia , Kärre Klas , Zappacosta Serafino
Published: March 2007

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from natural killer (NK) cell recognition in several systems. MHC class I gene products are released from the cell surface and can be found in sera as soluble forms. To investigate the possible immunoregulatory role of soluble HLA (sHLA) in NK cell‐target recognition, several sHLA antigens were studied for their ability to induce NK cell cytotoxicity modulation. NK cell‐target recognition was inhibited by the addition of sHLA during the cytotoxicity assay. Our results indicate that sHLA molecules can down‐regulate NK killing at the effector level. Moreover, different NK clones are able to specifically recognize different sHLA antigens. Kp43 molecules seem to be involved in the NK recognition of sHLA‐B7.
Effects of interferon‐α on cytokine profile, T cell receptor repertoire and peptide reactivity of human allergen‐specific T cells
Authors: Parronchi Paola , Mohapatra Subra , Sampognaro Salvatore , Giannarini Loretta , Wahn Ulrich , Chong Pele , Mohapatra Shyam , Maggi Enrico , Renz Harald , Romagnani Sergio
Published: March 2007

A large panel of T cell clones (TCC) specific for the recombinant form of Poa pratensis allergen (rKBG7.2 or Poa p9) were established from the peripheral blood of a grass pollen‐sensitive donor in the absence or presence of recombinant interferon‐α (IFN‐α) in bulk culture and their pattern of cytokine secretion, peptide reactivity and TCR Vβ repertoire was examined. The majority of allergen‐specific TCC derived in absence of IFN‐α produced high amounts of interleukin‐4 (IL‐4) and IL‐5 but not IFN‐γ (Th2 cells), while most of TCC derived in presence of IFN‐α produced IFN‐γ but not, or limited amounts of, IL‐4 and IL‐5 (Th1 or Th0 cells). Of 24 TCC established in the presence of IFN‐α, 22 were able to recognize a single allergen peptide, p26, while none of the clones established in the absence of IFN‐α showed a similar specificity. The majority of both clones expressed the Vβ2 element regardless of whether they were established in the presence of IFN‐α, but the presence of IFN‐α favored the expansion of Vβ2+, Vβ17+ and Vβ22+ Poa p9‐specific T cells, whereas in the absence of IFN‐α, other TCR Vβ‐bearing T cells (Vβ5, Vβ6.7 and Vβ14) were expanded in addition to Vβ2+ T cells. None of Vβ2+ clones established in the absence of IFN‐α reacted with p26, whereas all the Vβ2+ clones established in its presence responded to this peptide. IFN‐α also shifted the TCR Vβ repertoire of both Poa p9‐ and Lolium perenne group 1 (Lol p1)‐specific T cell lines generated from the same patient and from a different grass‐sensitive individual. These data demonstrate that IFN‐α modulates the development of allergen‐specific T cells in vitro, and suggest that IFN‐α may represent an useful tool for novel immunotherapeutic approaches in allergic disorders.
Quantitative analysis of multiple V‐region interactions among normal human IgG
Authors: Ayouba Ahidjo , Peltre Gabriel , Coutinho Antonio
Published: March 2007

There is to date no quantitative method for scoring putative V‐region interactions among serum antibodies, and the available qualitative techniques are not amenable to routine utilization. This deficit may explain the paucity of observations on the characteristics of the immune network, in contrast with the multiplicity of phenomenological descriptions on idiotype regulation.

We describe here a novel methodology that uses isoelectric focusing (IEF) to resolve human F(ab′)2 preparations from large pools of normal serum IgG into multiple bands, and computer‐aided data processing to analyze interactions between the resulting blotted proteins and normal serum IgG from individual donors. Our results show that in all normal human sera tested, there are IgG‐mediated interactions with a large number of IEF fractions of human F(ab′)2. These interactions are V region specific, as assessed by inhibition experiments and by the lack of binding of IgG monoclonal antibodies, and are characterized by average affinities that are in the micromolar range, as measured by surface plasmon resonance.
Epidermal growth factor can replace thymic mesenchyme in induction of embryonic thymus morphogenesis in vitro
Authors: Shinohara Takashi , Honjo Tasuku
Published: March 2007

The thymus is surrounded by a thin layer of mesenchyme and the epithelial‐mesenchymal interaction is known to be essential for the thymus development. To clarify the roles of mesenchyme in the thymus lobule formation that occurs around embryonic days 14–15 in vivo, we set up a three‐dimensional organ culture system. The epithelium of embryonic day 13 thymic primordium was separated from the mesenchyme and cultured in Matrigel (reconstituted basement membrane). Addition of the mesenchyme to a chamber separated by a membrane filter induced the lobule formation of the thymic epithelium in vitro. We found that epidermal growth factor (EGF) can replace the mesenchyme for lobulation of the embryonic thymus in vitro. Among other growth factors tested, only transforming growth factor (TGF)‐α was as effective as EGF, in agreement with the fact that EGF and TGF‐α bind to the same receptor. These results suggest that EGF or its family members may be involved in morphogenesis and differentiation of the thymus gland epithelium, although we cannot exclude the possibility that other unknown factors are required in vivo.
One cause for the apparent inability of human T cell clones to function as professional superantigen‐presenting cells is autoactivation
Authors: Nisini Roberto , Fattorossi Andrea , Ferlini Cristiano , D'Amelio Raffaele
Published: March 2007

Human T cell clones (TCC) are antigen‐presenting cells (APC) able to present peptides and superantigens (SAg) and to process and present intact proteins. TCC express major histocompatibility complex (MHC) class II antigens and molecules involved in the accessory signal delivery, such as B7.1 and B7.2/B70. Notwithstanding these observations, the role of professional APC has been often denied to T cells because anergy of responder T cells rather than proliferation has been observed following the TCC presentation in the absence of added professional APC. Here, we show that upon stimulation with free SAg, TCC undergo proliferative responses followed, after a 1‐week culture, by an SAg‐dependent unresponsiveness to T cell receptor (TCR)‐mediated stimuli, but not to interleukin‐2. The anergy induced by the SAg can not be prevented by the addition of autologous Epstein‐Barr virus (EBV)‐transformed B cells, indicating that the induction of anergy occurs also in the presence of conventional APC. Conversely, if the TCC are stimulated by SAg‐prepulsed irradiated APC, either EBV and TCC, the induction of anergy is not observed. After a 1‐week culture, in fact, TCC stimulated with APC‐bound SAg responded to TCR‐mediated stimuli, irrespective of the APC (EBV or TCC) used for the SAg presentation. Stimulation of TCC with free SAg in a semisolid medium that prevents T‐T cell contacts resulted in an activation followed by a state of anergy, suggesting that anergy is the consequence of SAg recognition at the single T cell level. These data indicate that the anergy observed in TCC upon a 1‐week culture in the presence of soluble SAg is not the result of an inherent inability of TCC to act as professional APC. Rather the phenomenon depends on the presence of soluble SAg, leading to T cell autostimulation.
T cell receptor‐γδ‐expressing fetal mouse thymocytes are generated without T cell receptor Vβ selection
Authors: Mertsching Elisabeth , Ceredig Rhodri
Published: March 2007

We investigated whether fetal mouse T cell receptor (TCR) γδ cells have been subjected to so‐called TCRβ selection at the CD25 stage of thymus development. To this end, we carried out a comparative three‐color flow microfluorimetric analysis of TCRβδ cells developing in the fetal, neonatal and adult thymus using monoclonal antibodies to CD2, CD8, CD24, CD25 and CD44. Day‐15 fetal TCRγδ cells were CD2+, suggesting an origin at a post‐CD25 stage. Molecular analysis of TCRβ rearrangements were also carried out. Thus, by semi‐quantitative polymerase chain reaction (PCR) amplification of Vβ6 and Vβ8 to Jβ2 rearrangements day‐15 fetal TCRγδ showed extensive TCRβ rearrangements, a finding confirmed by PCR amplification from single micromanipulated cells. Finally, sequencing analysis of 104 PCR‐amplified TCR VDJβ2 fragments showed that the majority (58%) were rearranged out of frame. Taken together, these phenotypic and molecular analyses suggest that fetal TCRγδ cells have not been subject to TCRβ selection.
CD31‐triggered rearrangement of the actin cytoskeleton in human natural killer cells
Authors: Poggi Alessandro , Panzeri Maria Carla , Moretta Lorenzo , Zocchi Maria Raffaella
Published: March 2007

In this report, we analyze whether CD31, also known as platelet‐endothelial cell adhesion molecule‐1 (PECAM‐1), can transduce an outside‐in signal in human natural killer (NK) lymphocytes in vitro. We show that CD31, but not HLA class‐I cross‐linking triggers an outside‐in transmembrane signal in NK lymphocytes, mediating cell spreading and cytoskeletal rearrangement. These phenomena are Mg2+, but not Ca2+ dependent, suggesting that signal transduction elicited by CD31 cross‐linking may involve an associated integrin. Two possible candidates would be αv and αL, whose function is known to depend on Mg2+. However, the CD31‐induced cytoskeletal rearrangement was not reduced by the use of αv‐ or αL‐specific F(ab′)2, suggesting that CD31 could transduce a signal by itself or by association with a still‐undefined integrin. Moreover, talin, but not vinculin or tubulin, appears to co‐localize with actin microfilaments in the membrane ruffles of NK cells that undergo cytoskeleton rearrangement following CD31 cross‐linking. Both spreading and cytoskeletal rearrangement appear to be regulated by intracellular cyclic‐3′,5′‐adenosine monophosphate (cAMP). Indeed, the activator of the adenylyl cyclase, forskolin, inhibited cell spreading and cytoskeletal rearrangement induced by CD31 cross‐linking. This phenomenon was also observed using the membrane‐permeants cAMP analog Sp adenosine‐3′,5′‐cyclic monophosphothioate (Sp‐cAMPS), but not its inactive isomer Rp‐cAMPS. Likewise, adhesion of NK lymphocytes to NIH/3T3 murine fibroblasts transfected with the cDNA encoding human CD31 was blocked by increasing intracellular cAMP levels. We suggest that intracellular cAMP may be involved in CD31‐mediated signal transduction, and may regulate NK‐endothelial cell adhesion and possibly, the tissue localization of NK cells.
Translatable immunoglobulin germ‐line transcript
Authors: Bachl Jürgen , Turck Christoph W. , Wabl Matthias
Published: March 2007

During B cell differentiation, the functional genes encoding immunoglobulin (Ig) heavy (H) and light (L) chains are generated by two rearrangement processes ‐ VDJ rearrangement generates the exon encoding the Ig variable (V) regions, and the class switch reconstructs a rearranged Ig H gene by exchanging the segment encoding the constant (C) region, which determines the Ig class. Both types of rearrangement are preceded by transcripts originating from a transcriptional start site 5′ of the I exon, which is then spliced to the C exons. These germ‐line transcripts, which are thought to be necessary for the initiation of both types of rearrangement, are said to be sterile. We demonstrate here that the μ germ‐line transcript is translatable into a polypeptide chain, to which we assign the symbol ψ. Thus, protein products of these transcripts might be part of or signal to the recombinases that catalyze Ig gene rearrangement.
JNK, but not MAPK, activation is associated with Fas‐mediated apoptosis in human T cells
Authors: Wilson David J. , Fortner Karen A. , Lynch David H. , Mattingly Raymond R. , Macara Ian G. , Posada James A. , Budd Ralph C.
Published: March 2007

Fas is a cell surface molecule that is expressed on a wide array of cell types and triggers apoptosis. While in most situations Fas ligation activates programmed cell death, on resting T lymphocytes it can co‐stimulate proliferation with the T cell receptor (TCR)/CD3 complex. This incongruity suggests that Fas may elicit signaling events that overlap with those used by proliferation cues. We observe that in the human T cell line Jurkat and in human peripheral blood lymphocytes, Fas stimulation does not signal by the Ras/Raf‐1/mitogen‐activated protein kinase (MAPK) pathway or by increased intracellular calcium. Rather, Fas ligation strongly activates Jun kinase (JNK). This activity, as well as Fas‐induced apoptosis, is blocked by increased levels of cAMP. The balance between proliferation and apoptosis by Fas triggering of T lymphocytes may therefore reflect a signaling ratio between TCR activation of the Ras/Raf‐1/MAPK pathway versus JNK activation by Fas.
Soluble CD40 ligand induces expression of CD25 and CD23 in resting human tonsillar B lymphocytes
Authors: Burlinson Ellen L. , Graber Pierre , Bonnefoy Jean‐Yves , Ozanne Bradford W. , Cushley William
Published: March 2007

In this report, we describe the dose‐dependent increase in both CD25 and CD23 levels on resting human B cells in response to CD40 ligation, as mediated by soluble CD40 ligand (sCD40L) or anti‐CD40 antibody. In combination with interleukin (IL)‐4, sCD40L had limited additive effects on CD25 expression, but significantly enhanced CD23 expression on tonsillar B cells. Interferon‐γ (IFN‐γ) exerted no inhibitory effect upon increases in CD25 or CD23 driven by CD40 ligation with sCD40L or anti‐CD40 antibody. These data suggest that the induction of CD25 and CD23 genes by IL‐4 is mediated, at least in part, by an IFN‐γ‐sensitive component, whereas gene activation driven via CD40 ligation involves signaling pathways which are not sensitive to IFN‐γ.
Inhibition of interleukin‐6 synthesis in an animal model of septic shock by anti‐C5a monoclonal antibodies
Authors: Höpken Uta , Mohr Michael , Strüber Andreas , Montz Hildegard , Burchardi Hilmar , Götze Otto , Oppermann Martin
Published: March 2007

The complement activation fragment C5a was recently shown to induce interleukin (IL)‐6 synthesis by peripheral blood mononuclear cells. To understand better the role of C5a in cytokine regulation in vivo, we investigated the effects of complement depletion by cobra venom factor (CVF) or of anti‐C5a monoclonal antibodies (mAb) on IL‐6 generation in an animal model of septic shock. Complement‐depleted pigs which were subsequently challenged with Escherichia coli generated significantly (p < 0.05) less IL‐6 during the 6‐h observation period than complement‐sufficient controls. To address specifically the role of C5a in IL‐6 regulation, we produced a C5a(57–74) peptide‐specific mAb (T13/9) which neutralizes the bioactivity of porcine C5a. The mAb T13/9 does not crossreact with the precursor protein C5. The pretreatment of pigs with anti‐C5a mAb T13/9 prior to the induction of sepsis resulted in a decrease of over 75 % in serum IL‐6 bioactivity compared to control animals (p < 0.0001). These results indicate a role for C5a in the modulation of IL‐6 synthesis in Gram‐negative bacteremia.
Characterization of an epitope of the human cytomegalovirus protein IE1 recognized by a CD4+ T cell clone
Authors: Gautier Nicolas , Chavant Emmanuelle , Prieur Eric , Monsarrat Bernard , Mazarguil Honoré , Davrinche Christian , Gairin Jean Edouard , Davignon Jean‐Luc
Published: March 2007

CD4+ T cells specific for human cytomegalovirus (HCMV) IE1 protein are potential effectors of the control of HCMV infection through cytokine production. Better knowledge of major histocompatibility complex (MHC)‐peptide‐T cell receptor (TcR) interactions in the CD4+ T cell response should result in a better design of immunizing peptides and is a prerequisite for the development of vaccines or anti‐cytomegalovirus therapy. In this study, the recombinant protein comprising residues 86–491 encoded by exon 4 of IE1 (GST‐e4) was cleaved by enzymatic digestion and analyzed by high pressure liquid chromatographymass spectroscopy (HPLC‐MS). We identified the 14‐residue epitope 162‐DKREMWMACIKELH‐175 recognized by an HLA‐DR8‐restricted clone, BeA3. Synthetic elongated, truncated and di‐Ala‐substituted peptides of the 18‐mer IE1 158‐IVPEDKREMWMACIKELH‐175 sequene were used to analyze the amino acid motifs involved in binding to HLA‐DR8 and recognition by the BeA3 clone. Substitutions which abolished (MW → AA), or decreased (RE → AA and MA → AA) T cell clone proliferation, cytokine production and cytotoxicity were identified. Loss of T cell function induced by the MW → AA substitution was associated with poor HLA‐DR8 binding. Decreased T cell function (RE → AA and MA → AA) was associated with good HLA‐DR8 binding, which suggested that these motifs were involved in TcR binding. Other substitutions induced potentiation of the T cell clone response: the IV → AA substitution induced stronger proliferation, but equivalent cytokine production, when compared with the reference peptide IE1 (158–175). CI → AA substitution induced strong potentiation of HLA‐DR8 binding, proliferation and interferon‐γ and interleukin‐4 production, possibly due to the removal of negative effects of Cys, Ile, or both side chains. Cytotoxicity was not improved by any substitution. Our results show modulation of the CD4+ T cell response according to the peptide residues involved in the HLA‐DR8‐peptide‐TcR interaction.
Dissociation between cytokine mRNA expression and protein production in shigellosis
Authors: Raqib Rubhana , Ljungdahl Åke , Lindberg Alf A. , Wretlind Bengt , Andersson Ulf , Andersson Jan
Published: March 2007

In our study, infection with Shigella dysenteriae type 1 (n = 16) or Shigella flexneri in adults (n = 5) was associated with a gradual accumulation of mRNA for interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, IL‐6, transforming growth factor‐β, IL‐10, IL‐4, TNF‐β, interferon (IFN)‐γ and perforin in the rectal biopsy samples during the convalescent stage of the disease demonstrated by in situ hybridization. In contrast, immunohistochemical staining in rectal tissues of cytokine protein‐producing cells at the single‐cell level exhibited a steady‐state expression during 2–36 days after the onset of the disease. The frequency of cytokine mRNA‐expressing cells varied in the range of 3–100‐fold higher than that of the corresponding protein‐synthesizing cells. The accumulation of cytokine mRNA in vivo during shigellosis represented a long‐lasting phenomenon throughout the disease course, and may be linked to its immunopathogenesis. The results also indicate that assessment of both protein and mRNA in vivo may provide complementary information. Stimulation in vitro of peripheral blood mononuclear cells from normal healthy donors with Shigella‐derived lipopoly‐saccharide or shiga toxin was carried out to elucidate the role of Shigella antigens in the regulation of translation of cytokine‐specific mRNA. The incidence of cytokine (IFN‐γ, IL‐6 and TNF‐α) mRNA‐ and cytokine protein‐expressing cells was very similar and congruent after both these Shigella‐derived stimuli. We could, thus, not find evidence for shiga toxin‐induced down‐regulation of cytokine mRNA translation as the explanation for the observed discrepancy between cytokine mRNA and protein levels in the tissue biopsies.
Human dendritic cells activate T lymphocytes via a CD40: CD40 ligand‐dependent pathway
Authors: McLellan Alexander D. , Sorg Rüdiger V. , Williams Lisa A. , Hart Derek N. J.
Published: March 2007

The CD40: CD40 ligand (CD40L) interaction provides T lymphocyte‐mediated help for B lymphocyte and monocyte function but has also been shown to serve as a co‐stimulus for T lymphocyte activation. In this report, we studied the regulation of CD40 expression and its functional relevance for the human dendritic cell (DC) stimulation of T lymphocytes. Only a small subpopulation of directly isolated blood DC expressed CD40. However, CD40 was rapidly up‐regulated by culture, and its expression was further enhanced by interleukin (IL)‐1α, IL‐1β, IL‐3, tumor necrosis factor‐α and granulocyte/macrophage‐colony‐stimulating factor. Expression of CD40L on DC was not detected. The proliferation of T lymphocytes in an allogeneic mixed leukocyte reaction, stimulated by blood DC or epidermal Langerhans cells, was significantly reduced in the presence of the CD40 immunoglobulin (CD40Ig) fusion protein or CD40L monoclonal antibodies. Cross‐linking of CD40 on directly isolated DC with mouse CD40L trimer (mCD40LT) markedly augmented CD80 and CD86 up‐regulation. Nevertheless, the same cross‐linking mCD40LT inhibited DC stimulated T lymphocyte proliferation. When CD40Ig was added simultaneously with CTLA‐4Ig, only minimal and variable additional inhibition of DC‐stimulated allogeneic T lymphocyte proliferation and IL‐2 secretion was observed, compared to each fusion protein alone. These results suggest that both CD80/CD86‐dependent and ‐independent components of DC‐T lymphocyte CD40: CD40L co‐stimulation exist and further emphasize that the majority of blood DC have to differentiate or be activated to express co‐stimulatory molecules.
Death of germinal center B cells without DNA fragmentation
Authors: Nakamura Masanori , Yagi Hideki , Kayaba Shoichi , Ishii Tadashi , Gotoh Takahiro , Ohtsu Susumu , Itoh Tsunetoshi
Published: March 2007

During the selection of B cells within germinal centers (GC) on the basis of their affinity for T‐dependent antigen, B cells not positively selected are eliminated within GC. This process of B cell death has been considered to be apoptosis. In a recent study, we have reported that, although a substantial number of thymocytes were considered to be dead because of their extremely small cell size and heavy chromatin condensation even though they were not yet phagocytosed (pyknosis), they were devoid of DNA fragmentation, the most characteristic feature for apoptosis. In this study, we examined in vivo the mechanism of B cell death within GC by using the terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP‐biotin nick end‐labeling (TUNEL) method to detect DNA double‐strand breaks. TUNEL+ B cells were scattered throughout the upper dark and the light zones of GC. Double staining of the sections by the TUNEL method and acid phosphatase (AcP) activities showed that all the TUNEL+ B cells were phagocytosed by macrophages. Light microscopic and ultrastructural studies revealed the presence of small unphagocytosed B cells within the light zone. These cells are undoubtedly dead because they were much smaller than surrounding lymphoid cells and have a heavy chromatin condensation. Furthermore, ultrastructural detection of DNA fragmentation confirmed that these small unphagocytosed B cells were TUNEL−, implying that DNA fragmentation is not primarily involved in the cell death process of these small dead B cells. These results indicate that most B cells, not positively selected and thus destined to be eliminated, die within GC without DNA fragmentation, and are subsequently phagocytosed by macrophages and become TUNEL+. Typical apoptosis, characterized by DNA fragmentation in situ, is not the predominant type of cell death that occurs during the selection of B cells in GC.
CD28‐dependent killing by human YT cells requires phosphatidylinositol 3‐kinase activation
Authors: Lu Yiling , Rodriguez Ron , Bjorndahl Jay , Phillips Catherine A. , Trevillyan James M.
Published: March 2007

CD28/B7 interactions have been demonstrated to provide a co‐stimulatory signal for the generation of CD8+ cytotoxic T lymphocytes in the absence of CD4+ T helper cells. The CD28 signals required for induction of cytotoxicity have yet to be described. To investigate further the biochemical signaling pathways associated with CD28‐dependent cytotoxicity, we have studied the human thymic leukemia cell line, YT. YT cells kill B7+ targets in a non‐major histocompatibility complex (MHC)‐restricted, CD28‐dependent manner. CD28 ligation on the surface of YT cells caused a rapid increase in the tyrosine phosphorylation of four major cellular substrates with masses estimated to be 110, 95, 85, and 44 kDa. The 110 and 85 kDa substrates were identified as the catalytic and regulatory subunits, respectively, of phosphatidylinositol 3‐kinase (PI3‐K). Engagement of CD28 caused the rapid receptor association and activation of PI3‐K but did not activate phospholipase Cγ. CD28‐induced tyrosine phosphorylation and PI3‐K activation was independent of p56lck protein tyrosine kinase (PTK) activity (previously reported to be associated with CD28) and was insensitive to inhibition by the PTK inhibitor herbimycin A. Two structurally and mechanistically dissimilar inhibitors of PI3‐K, wortmannin and 2‐(4‐morpholinyl)‐8‐phenyl‐4H‐1‐benzopyran‐4‐one (LY294002) also failed to block CD28‐dependent tyrosine phosphorylation events or the association of PI3‐K with the CD28 receptor. However, both drugs inhibited CD28‐dependent cytotoxicity and CD28 receptor associated PI3‐K activity with IC50 values similar to the reported IC50 values for PI3‐K inhibition. Although herbimycin A did not significantly block the observed CD28‐dependent tyrosine phosphorylation or PI3‐K activation, herbimycin did block CD28‐dependent cytotoxicity in a dose‐dependent manner. These data support a role for PI3‐K activation in the CD28‐dependent initiation of cytotoxic effector function and suggest that a herbimycin sensitive step(s) is either CD28‐independent, resides within a PI3‐K‐independent CD28 signaling pathway, or is downstream of CD28‐dependent PI3‐K activation.
Immune complex‐induced interleukin‐6, interleukin‐10 and prostaglandin secretion by human monocytes: a network of pro‐ and anti‐inflammatory cytokines dependent on the antigen: antibody ratio
Authors: Berger Stefan , Balló Hilmar , Stutte Hans Jochen
Published: March 2007

We have used two experimental models of immune complexes to study the secretion of interleukin (IL)‐10, IL‐6 and their connection with the immune complex‐induced synthesis of prostaglandin (PG) E2 by human monocytes in vitro. Immune complexes formed of tetanus toxoid and polyclonal anti‐tetanus toxoid antiserum as well as heat‐aggregated human serum immunoglobulins induced the release of IL‐6 and IL‐10 in a dose‐ and antigen: antibody ratio‐dependent manner. Antigen‐antibody complexes formed near equivalence were most effective in induction of a cytokine response. PGE2 could augment the immune complex‐induced IL‐6 and IL‐10 secretion, but alone, did not induce cytokine secretion. IL‐10 was capable of down‐regulating the release of IL‐6 and PGE2. Additionally, we demonstrated that endogenously synthesized IL‐10 limited the immune complex‐induced secretion of proinflammatory cytokines tumor necrosis factor‐α and IL‐1β. All three regulatory factors examined here share anti‐inflammatory properties and are closely associated with the T helper type 2 (Th2) immune response. We conclude that immune complexes, besides their well‐known ability no cause acute and chronic inflammation, can mediate immunosuppressive effects and influence the balance of Th1/Th2 responses.
Affinity maturation and hypermutation in a simulation of the humoral immune response
Authors: Celada Franco , Seiden Philip E.
Published: March 2007

By experimenting with a cellular automaton model of the immune system, we have reproduced affinity maturation of the antibody response, a somatic adaptation to a changing environment. The simulation allowed the isolation of a number of variables, e.g. the fraction of repertoire available, the magnitude of the change in affinity with mutation, the mutation frequency and its focus on the complementarity‐determining regions (CDR) of the antibody. Multiple series of immunizations were run in machina where the contribution of each variable was evaluated against the maturation observed. We found that hypermutation is not necessary for affinity maturation if the repertoire of B cell specificities is sufficiently complete, but is essential when the B cell diversity is limited (which happens to be the case in vivo), as it fills the holes in the repertoire and allows selection by antigen. Maturation also depends on the magnitude of the change in affinity with mutation, and we supply some necessary limits on this parameter. For mutations confined to the CDR, the most efficient maturation occurs at mutation rates of 0.2 per paratope and per cell division. When mutations also affect the framework regions, the peak of the most effective CDR mutation rate moves progressively to lower values. A most sensitive parameter is the speed of maturation, which reflects the rate of expansion of mutated clones. Comparing it with biological observations can help to discriminate between alternative hypotheses on the phenomena of hypermutation and affinity.
Evidence of a T helper type 2 activation in human schistosomiasis
Authors: Araújo Maria Ilma , de Jesus Amélia Ribeiro , Bacellar Olívia , Sabin Elizabeth , Pearce Edward , Carvalho Edgar M.
Published: March 2007

The lymphocyte proliferative response and cytokine production to S. mansoni antigen in vitro were evaluated in 22 schistosomiasis patients living in an area endemic for this disease. The majority of patients (86%) showed no lymphocyte proliferative response and none of them showed interferon‐γ (IFN‐γ) production, following in vitro stimulation with soluble adult worm antigen preparation (SWAP). In contrast, interleukin (IL)‐5 (2038 ± 1757 pg/ml) and IL‐10 (867 ± 762 pg/ml) were detected in peripheral blood mononuclear cell (PBMC) cultures stimulated with SWAP. Moreover, mRNA for IL‐4 was detected in SWAP‐stimulated PBMC from 4 of 6 patients evaluated. Restoration of lymphoproliferative response was achieved in 4 of 6 patients by adding anti‐IL‐10 monoclonal antibody (mAb) to PBMC cultures [mean stimulation index (SI) in the presence of antigen = 2.7 ± 2.9; SI in the presence of antigen plus anti‐IL‐10, 21 ± 16]. Restoration of IFN‐γ production by addition of anti‐IL‐10 mAb was achieved in 4 of 12 patients evaluated (248, 350, 687 and 710 pg/ml). Moreover, the addition of IL‐10 to PBMC cultures of 3 schistosomiasis patients and 2 cured subjects who had high lymphoproliferative responses to SWAP resulted in the suppression of these responses by 90%, and completely suppressed IFN‐γ production in one of the subjects, whose PBMC produced IFN‐γ after stimulation with SWAP. The presence of IL‐4 mRNA, high levels of IL‐5, and the absence of IFN‐γ in PBMC culture supernatants from infected patients, supports the conclusion that patients living in an endemic area of schistosomiasis express a predominant T helper type 2 response. The high levels of IL‐10 and the ability of neutralizing anti‐IL‐10 mAb to restore T cell responses indicate that this cytokine plays an important role in the modulation of T cell responses in schistosomiasis.
Mercury induces polyclonal B cell activation, autoantibody production and renal immune complex deposits in young (NZB × NZW)F1 hybrids
Authors: Al‐Balaghi Sanad , Möller Erna , Möller Göran , Abedi‐Valugerdi Manuchehr
Published: March 2007

It is well established that in susceptible mouse strains, chronic treatment with subtoxic doses of mercuric chloride (HgCl2) induces a systemic autoimmune disease, which is characterized by increased serum levels of IgG1 and IgE antibodies, by the production of anti‐nucleolar antibodies and by the development of immune complex‐mediated glomerulonephritis. Susceptibility to mercury is partly under the control of major histocompatibility complex genes. To study the susceptibility to mercury further, we investigated the in vivo effects of mercury in young autoimmune disease prone (NZB × NZW)F1 (H‐2d/z) mice prior to establishment of spontaneous autoimmune disease. Mercury‐susceptible SJL (H‐2s) mice and mercury low‐responder BALB/c (H‐2d) mice were used as positive and negative controls, respectively. In (NZB × NZW)F1 mice, treatment with mercury stimulated an intense antibody formation characterized by increased numbers of splenic IgG1 and IgG3 antibody‐producing cells as well as by elevated serum IgE levels. Injection with mercury also induced an increased production of IgG1, IgG2b and IgE antibodies in SJL, but not in BALB/c mice. The mercury‐induced IgG1 response in (NZB × NZW)F1 and SJL mice was found to be polyclonal and autoantibodies against double‐stranded (ds)DNA, IgG, collagen, cardiolipin, phosphatidylethanolamine as well as antibodies against the hapten trinitrophenol were produced. In addition, SJL, but not (NZB × NZW)F1 or BALB/c mice, produced IgG1 anti‐nucleolar antibodies after treatment with mercury. Further studies demonstrated that (NZB × NZW)F1 and SJL mice developed high titers of renal mesangial immune complex deposits containing IgG1 antibodies 3 weeks after injection with mercury. Thus, a mouse strain genetically prone to develop spontaneous autoimmune diseases is highly susceptible to mercury‐induced immunopathological alterations.
CD40‐mediated regulation of interleukin‐4 signaling pathways in B lymphocytes
Authors: Siepmann Kirsten , Wohlleben Gisela , Gray David
Published: March 2007

The importance of cytokines in controlling immunoglobulin isotype switching is well known. Given the defect in switching to IgG, IgA and IgE isotypes in mice and humans that carry mutations in the CD40 and CD40 ligand genes, we have investigated the role of CD40 ligation in controlling B cell responses to interleukin (IL)‐4. We have found that CD40‐mediated signals cause a fivefold up‐regulation of IL‐4 receptor (IL‐4R) on the B cell surface and that this is associated with a 100–1000‐fold increase in the cells' responsiveness to the cytokine. While we found no evidence of increased affinity or structural change of the receptor, we do find that prestimulation of B cells with anti‐CD40 antibodies brings about several changes in the IL‐4 signaling pathways. Subsequent delivery of IL‐4 to CD40‐prestimulated cells provokes intracellular signals distinct from those induced in resting B cells in response to IL‐4. While resting B cells phosphorylate Jak3 kinase shortly after IL‐4 activation, cells pre‐incubated with anti‐CD40 exhibit active dephosphorylation of this molecule and phosphorylation of proteins of around 45 kDa upon addition of IL‐4. The common γ chain, Jak3 and Jak1 can all be immunoprecipitated in normal amounts with the IL‐4R chain after CD40 prestimulation. We show that the observed dephosphorylation of Jak3 may be due to a stable association with the src‐homology protein tyrosine phosphatase SH‐PTP2. In contrast, the enzyme appears to be inactive and to dissociate very quickly from the signaling complex in cells that are stimulated with IL‐4 alone.