bluuebunny/crossref_metadata_2025 huggingface dataset:
Central tolerance: Clonal deletion or clonal arrest?
Authors: Swat Wojciech , Boehmer Harald Von , Kisielow Pawel
Published: March 2007

Studies in various experimental animals have shown that developing T cells with specificity for self antigens can be prevented from maturation at an early stage of development. While several in vitro and in vivo experiments have shown that the mechanism of silencing autospecific T cells is the deletion of immature CD4+8+ thymocytes other experiments were interpreted to indicate that tolerance could also result from developmental arrest of more immature CD4−8+ thymocytes not involving cell death. Here we show that immature CD4−8+ cells when confronted with T cell receptor ligands in vitro neither survive nor differentiate into cells which cannot be deleted, indicating that clonal elimination rather than developmental arrest is the mechanism of central tolerance of all immature T cells.
Delay in expression of a mammary tumor provirus is responsible for defective clonal deletion during postnatal period
Authors: Niimi Naoya , Tomida Shin , Ueda Minoru , Kaneda Toshio , Ando Yoshihiro , Takeuchi Makoto , Yoshikai Yasunobu , Wajjwalku Worawidh
Published: March 2007

A gene‐encoding ligand for deletion of T cells bearing TcRVβ6 and Vβ8.1 cosegregates a new mammary tumor provirus locus, Mtv‐50 in NC mice. The sequence of the open reading frame (ORF) in the 3′ long terminal repeat (LTR) of Mtv‐50 was strikingly similar to those of Mtv‐7, Mtv‐43 and exogenous mouse mammary tumor virus (SW) with properties of minor lymphocyte stimulating antigen 1a. Consistent with previous reports, clonal deletion of mature thymocytes bearing TcRVβ6 was defective during the early postnatal period of mice carrying Mtv‐50. Appreciable levels of mRNA corresponding to common Mtv ORF and Mtv‐6 ORF were expressed in the neonatal thymus, while little, if any, mRNA corresponding to Mtv‐50 ORF was detected in the thymus at the early postnatal stage. Delay in expression of Mtv‐50 ORF during the postnatal period may be responsible for the failure of clonal deletion of Vβ6‐T cells in the early postnatal life of mice carrying Mtv‐50.
Helper effector function of human T cells stimulated by anti‐CD3 mAb can be enhanced by co‐stimulatory signals and is partially dependent on CD40‐CD40 ligand interaction
Authors: Kwekkeboom Jaap , De Rijk Diana , Kasran Ahmad , Barcy Serge , De Groot Cornelis , De Boer Mark
Published: March 2007

In this study we have investigated whether anti‐CD3‐induced human T cell help for immunoglobulin production could be enhanced by co‐stimulation of the T cells via other T cell surface molecules, and the contribution of CD40‐CD40 ligand interaction to the execution of T helper effector function induced by these different stimulatory signals. In a system in which irradiated tonsillar T cells were stimulated with immobilized anti‐CD3 monoclonal antibody (mAb), it was found that ligation of CD2 with a mitogenic pair of mAb considerably enhanced anti‐CD3‐induced T cell help for immunoglobulin production. Likewise, ligation of CD28 with mAb enhanced T helper activity, although to a lesser extent. Upon addition of anti‐CD28 and anti‐CD2 mAb together, an even higher immunoglobulin production was observed. This combination resulted in a four‐ to fivefold increase in immunoglobulin production as compared to cultures in which T cells were stimulated with anti‐CD3 mAb alone. The effect of ligation with B7, the natural ligand of CD28, was studied in a system which utilizes the presentation of anti‐CD3 mAb on human FcγRII‐expressing mouse fibroblasts which were co‐transfected with human B7. It appeared that B7 could stimulate help for immunoglobulin production much more efficiently than ligation of CD28 with mAb did. Physical separation of B cells from T cells led to complete abrogation of immunoglobulin production. Blocking of CD40 with specific mAb, which have no intrinsic B cell stimulatory properties, or the CD40 ligand with a soluble CD40‐human IgM fusion protein, resulted in dose‐dependent, but only partial, inhibition of T cell‐dependent immunoglobulin production with all modes of T cell activation tested. A clear correlation was found between the induction of CD40 ligand expression on the T cells by the different modes of co‐stimulation and subsequent immunoglobulin production by the B cells. It is concluded that ligation of CD28 and/or CTLA‐4, and of CD2 can generate co‐stimulatory signals for T cell help for immunoglobulin production, which was found to be only partially dependent on the CD40‐CD40 ligand interaction.
Interleukin‐3 and Bcl‐2 cooperatively inhibit etoposide‐induced apoptosis in a murine pre‐B cell line
Authors: Ascaso Rosalia , Marvel Jacqueline , Collins Mary K. L. , López‐Rivas Abelardo
Published: March 2007

Murine bone marrow‐derived hemopoietic cells, dependent on interleukin (IL)‐3 for their growth in culture, undergo programmed cell death, or apoptosis, upon cytokine withdrawal. The topoisomerase II inhibitor etoposide causes a more rapid onset of apoptosis in the IL‐3‐dependent cell line BAF3, deprived of IL‐3. This acceleration of apoptosis by etoposide is prevented by inhibitors of RNA and protein synthesis and by the nucleases inhibitor aurintricarboxylic acid. The presence of IL‐3 or overexpression of the oncogene bcl‐2 caused a marked delay in the induction of apoptosis by etoposide, acting in a cooperative manner. The time at which the apoptotic program is irreversible is close to the induction of endonuclease activity as indicated by the effect of the delayed addition of either IL‐3 or aurintricarboxylic acid on the onset of apoptosis, suggesting the importance of endonuclease activation in the development of apoptosis in hemopoietic cells.
Absence of peripheral clonal deletion and anergy in immune responses of T cell‐reconstituted athymic mice
Authors: Williams Owen , Aroeira Luiz S. , Martínez‐A Carlos
Published: March 2007

Superantigens induce clonal deletion of reactive T cells in the thymus and clonal deletion and anergy in the periphery of euthymic mice. In this report we have assessed the ability of Staphylococcal enterotoxin B (SEB) to induce peripheral tolerance in nude mice reconstituted with normal, syngeneic T cells. Immunization of reconstituted nude mice with SEB resulted in lethal toxic shock in a large fraction of the animals. Such lethality was never observed in the normal donor mouse strain. Analysis of lymphokine production in response to SEB showed that reconstituted nude mice produced higher levels of interleukin‐2 and tumor necrosis factor‐α, but lower levels of interleukin‐4, than euthymic control mice. Furthermore, SEB was unable to promote either clonal elimination or induction of anergy in the SEB‐responsive peripheral T cells, despite the fact that reconstituted nude mice did produce high levels of corticosterone upon treatment with SEB. These results imply a lack of control over immune responses to superantigen in T cell‐reconstituted athymic mice.
Involvement of p21ras activation in T cell CD69 expression
Authors: d'Ambrosio Daniele , Cantrell Doreen A. , Frati Luigi , Santoni Angela , Testi Roberto
Published: March 2007

The involvement of p21ras in the induction of the early activation antigen CD69 was investigated in T cells. Expression of a v‐Ha‐ras coding for a constitutively active ras protein in Jurkat cells resulted in CD69 induction on the cell surface. Transfected ras was shown to be constitutively activated and functionally efficient, since it could be immunoprecipitated in the guanosine triphosphate (GTP)‐bound form and it induced transactivation of an AP‐1 consensus‐chloramphenicol acetyltransferase reporter gene. The requirement for ras activation in T cell receptor (TcR) CD3‐mediated CD69 induction was also investigated. The expression of a dominant negative c‐Ha‐ras‐N17 mutant markedly reduced the amount of GTP that could be immunoprecipitated from ras proteins after TcR/CD3 triggering in Jurkat cells, and concomitantly decreased TcR/CD3‐mediated CD69 induction. These results suggest a central role for ras in TcR/CD3‐mediated CD69 expression in T cells.
Effect of a selective protein kinase C inhibitor, Ro 31–8425, on Mac‐1 expression and adhesion of human neutrophils
Authors: Sullivan John A. , Merritt Janet E. , Budd John M. , Booth Robert F. G. , Hallam Trevor J.
Published: March 2007

The role of protein kinase C (PKC) in mediating up‐regulation of macrophage 1 adhesion protein (Mac‐1) and adhesion of neutrophils in response to physiological agonists is not clear. Previous studies have relied on use of phorbol esters to activate PKC directly or on results obtained with non‐selective inhibitors of protein kinases. 3‐[8‐(Aminomethyl)–6,7,8,9‐tetrahydropyridol[1,2‐a]‐indol‐10‐yl]‐4‐(1‐methyl‐3‐indolyl)‐1H‐pyrrole‐2,5‐dione hydrochloride (Ro 31–8425) is a potent and highly selective inhibitor of PKC (Bit et al. J. Med. Chem. 1993. 36: 21). In these studies Ro 31–8425 has been used to define, more definitively, the role of PKC in mediating complement fragment C5a (C5a)‐stimulated up‐regulation of Mac‐1 and adhesion of neutrophils to endothelial cells and to bovine serum albumin (BSA)‐coated plastic. Phorbol 12, 13 dibutyrate (PBu2) increased surface expression of Mac‐1 and stimulated adhesion of neutrophils to endothelial cells and to BSA‐coated plastic. This confirms previous reports that activation of PKC can stimulate these responses. The PKC inhibitor, Ro 31–8425, inhibited the PBu2‐stimulated responses, which confirms that Ro 31–8425 was effective in inhibiting PKC in these neutrophils. A more physiological agonist, C5a, also increased surface expression of Mac‐1 and adhesion of neutrophils to endothelial cells and BSA‐coated plastic. However, the responses to C5a were unaffected by Ro 31–8425. These results suggest that, although activation of PKC can promote up‐regulation of Mac‐1 and adhesion of neutrophils, this does not appear to be the physiological pathway. A non‐selective protein kinase inhibitor, staurosporine, inhibited both PBu2 and C5a‐stimulated adhesion. This suggests that a protein kinase other than PKC, possibly a tyrosine protein kinase, is likely to be involved in mediating C5a‐stimulated Mac‐1 up‐regulation and adhesion. These results emphasise the need for caution in interpreting experiments and assuming a role for PKC. Use of a potent and selective inhibitor of PKC, Ro 31–8425, provides more definitive information.
Extracellular matrix components of the mouse thymus microenvironment. IV. Modulation of thymic nurse cells by extracellular matrix ligands and receptors
Authors: Villa‐Verde DÉA Maria Serra , Machado Jussara , Lagrota‐Candido  , Vannier‐Santos Marcos Andre , Chammas Roger , Brentani Ricardo Renzo , Savino Wilson
Published: March 2007

Extracellular matrix (ECM) proteins can influence cell migration and differentiation in a variety of cell systems. Within the thymus, these molecules are heterogeneously distributed, and their physiological role is poorly understood. This prompted us to carry out in vitro studies using the thymic nurse cell (TNC) model. We observed that fibronectin and laminin accelerate spontaneous in vitro release of thymocytes from TNC, whereas anti‐ECM antibodies exhibited a blocking effect. Similar results were obtained with anti‐ECM receptor reagents. Moreover, these antibodies abrogated in vitro reconstitution of TNC complexes and thymocyte adhesion to TNC‐derived epithelial cultures.

Our results indicate that lymphocyte traffic in TNC (comprising both entrance into and exit from the epithelial structure) is affected by interactions involving extracellular matrix ligands and receptors. In this respect, the dynamic analysis of thymic nurse cell complexes should be regarded as a relevant in vitro tool for functional studies of distinct adhesion molecules in intrathymic lymphocyte traffic.
The internalization of the IgG2a antigen receptor does not require the association with Ig‐α and Ig‐β but the activation of protein tyrosine kinases does
Authors: Weiser Peter , Riesterer Christa , Reth Michael
Published: March 2007

The B cell antigen receptor of class IgM is a multimeric protein complex containing the membrane‐bound immunoglobulin molecule and a heterodimer of the two B cell‐specific transmembrane proteins Ig‐α and Ig‐β. The B cell antigen receptor fulfills a dual role on the surface of B cells. First, it is a signal transduction complex which can activate protein tyrosine kinases and induce the release of Ca2+ ions from intracellular stores. Second, its internalization mediates the specific uptake of bound antigens, which are processed intracellularly and presented as major histocompatibility complex‐bound peptides on the cell surface. In case of the IgM antigen receptor, the association with the heterodimer is necessary for expression of large amounts of IgM on the surface. We show here that the IgG2a antigen receptor can be expressed on the surface of myeloma cells in two structurally different forms: either with or without the Ig‐α/Ig‐β heterodimer. A functional comparison of the two forms of antigen receptors demonstrates that the Ig‐α and Ig‐β molecules are required for the activation of protein tyrosine kinases after cross‐linking of the B cell antigen receptor. In contrast, both forms of IgG2a are equally well internalized. This suggests that Ig‐α and Ig‐β are essential for signal transduction through the IgG2a antigen receptor, whereas internalization can occur independently of the heterodimer.
Kinetics of intestinal intraepithelial lymphocytes during acute graft‐versus‐host disease in mice
Authors: Tsuzuki Toyonori , Yoshikai Yasunobu , Ito Masafumi , Mori Naoyoshi , Ohbayashi Masaharu , Asai Junpei
Published: March 2007

We examined the kinetics of intestinal intraepithelial lymphocytes (IEL) and the incidence of apoptosis at villus or crypt sites during the development of non‐irradiated acute graft‐versus‐host disease (GVHD). The first IEL to increase were host‐origin on day 3 and the donor‐derived IEL appeared first on day 12 after GVHD induction. Unique CD3+CD4+CD8α/α+ IEL were significantly increased on day 6 and an appreciable number of IEL bearing T cell receptor Vβ capable of recognizing self‐superantigen were detected on day 9. The sudden appearance of apoptosis and reduction of mitotic activity occurred on day 12, accompanied by a dramatic decrease of CD3+CD4−CD8α/α+ IEL of host origin. CD8α/α+ IEL of host origin, which expand and then decrease by apoptosis at the early stage of acute GVHD, may be associated with pathogenesis of the enteropathy occurring during acute GVHD.
Heat‐stable antigen/CD24 on mouse T lymphocytes: evidence for a costimulatory function
Authors: Hubbe Marcus , Altevogt Peter
Published: March 2007

Heat‐stable antigen (HSA)/mouse CD24 (formerly termed Nectadrin) is a membrane glycoprotein with an unusual structure consisting of a small protein core and extensive glycosylation. It is expressed by hematopoietic cells but not by mature T lymphocytes. HSA on accessory cells is an important costimulatory molecule required for the clonal expansion of T lymphocytes. HSA is also involved in cell‐cell adhesion events and the isolated antigen has been shown to possess self‐binding properties. In the present study we have re‐investigated the role of HSA in T cell proliferation. We find that following stimulation of T lymphocytes with concanavalin A or of CD4+ T lymphocytes with a combination of anti‐CD3/CD28 monoclonal antibodies (mAb) the HSA antigen is transiently expressed. The expression correlated with the appearance of CD25 and a CD2 activation epitope at the cell surface. Induction of HSA was also seen in vivo on Vβ8+ T lymphocytes in BALB/c mice that were injected with Staphylococcal enterotoxin B. Biosynthetic labeling and analysis of mRNA by reverse transcriptase‐polymerase chain reaction showed that HSA was synthesized by activated T lymphocytes. A combination of anti‐CD3 and mAb 79 to HSA was incapable of inducing proliferation of purified CD4+ T lymphocytes. However, the antibody strongly enhanced the response obtained with a combination of anti‐CD3/CD28 mAb. The augmenting effect of the HSA‐specific mAb was dose dependent. Since HSA is bound to the membrane via a glycosyl‐phosphatidylinositol (GPI) anchor and GPI‐anchored molecules have been implicated in lymphocyte activation, it is conceivable that HSA is not only a costimulatory molecule on accessory cells but is also a signaling molecule in T lymphocytes. The possibility of a homotypic HSA/HSA interaction between T lymphocytes and accessory cells is discussed.
“Natural” presentation of human papillomavirus type‐16 E7 protein to immunocompetent mice results in antigen‐specific sensitization or sustained unresponsiveness
Authors: Chambers Mark A. , Wei Zhang , Coleman Nicholas , Nash Anthony A. , Stanley Margaret A.
Published: March 2007

We have used a mouse model that utilizes the exclusively epithelial nature of human papillomavirus (HPV) infections to investigate the in vivo immune response to the E7 protein of human papillomavirus type‐16. A keratinocyte cell line expressing E7 protein has been established and grafted onto syngeneic mice using a transplantation technique that permits the reformation of a differentiated epithelium on a granulation tissue bed. In this way viral antigens may be presented to the immune system in a way comparable to natural infection. A delayed‐type hypersensitivity (DTH) response was studied post grafting by intradermal challenge with recombinant E7 protein. A significant response to E7 has been demonstrated in this way; however, priming with a low amount of HPV‐16 E7 antigen induces immunological unresponsiveness, as measured by a loss of DTH reactivity to the protein, and persistence of keratinocytes expressing E7. Lymphocytes from mice exhibiting DTH reactivity have been shown to proliferate when stimulated with purified recombinant E7 protein in vitro, while immunoperoxidase staining from the sites of immunologically‐induced inflammation has defined the cell infiltrate to be phenotypically characteristic of DTH. The observations reported here have important implications for vaccine strategy.
Leishmania major‐specific CD8+ T cells are inducers and targets of nitric oxide produced by parasitized macrophages
Authors: Stefani Mariane M. A. , Müller Ingrid , Louis Jacques A.
Published: March 2007

Lines of Leishmania major‐specific CD8+ T cells were derived from the lymph nodes and spleens of CBA mice, immune following resolution of a primary infection, 7 days after secondary challenge with viable L. major. Specific stimulation of these CD8+ T cells by bone marrow‐derived macrophages infected with L. major led to the release of interferon‐γ by CD8+ T cells and nitric oxide by macrophages. Interestingly, the nitric oxide released by bone marrow‐derived macrophages down‐regulated the production of interferon‐γ by specifically activated CD8+ T cells. The proliferation and long‐term maintenance of these parasite‐specific CD8+ T cells was impaired by the nitric oxide produced by stimulating infected macrophages as a result of cytokines released by activated CD8+ T cells. Taken together, the results indicate that L. major‐specific CD8+ T cells are sensitive to the toxic effect of the nitric oxide that they induce.
Differential expression of APO‐1 on human thymocytes: Implications for negative selection?
Authors: Debatin Klaus‐Michael , Süss Dorothee , Krammer Peter H.
Published: March 2007

Negative selection during T cell ontogeny involves selective induction of apoptosis in thymocytes. In peripheral lymphoid cells, apoptosis may be mediated via the APO‐1 pathway. Here we report that APO‐1 is constitutively expressed on the vast majority of human thymocytes but down‐regulated at a mature stage of thymocyte development (TCRhi). This stage of development is characterized by CD28hi, CD44hi, CD69hi and up‐regulation of Bcl‐2 protein. We define a new thymocyte subpopulation that expresses high levels of APO‐1 and intermediate levels of T cell receptor α/β (TCRim/APO‐1hi). The TCRim/APO‐1hi population contains a large fraction of dead cells, suggesting that the APO‐1 pathway may be involved in negative selection of at least a fraction of thymocytes after intrathymic activation.
Two tyrosinase nonapeptides recognized on HLA‐A2 melanomas by autologous cytolytic T lymphocytes
Authors: Wölfel Thomas , Van Pel Aline , Brichard Vincent , Schneider Jörg , Seliger Barbara , Zum Büschenfelde Karl‐Hermann Meyer , Boon Thierry
Published: March 2007

A number of cytolytic T lymphocyte (CTL) clones derived from several melanoma patients have been found to recognize a majority of melanomas from HLA‐A2 patients. We have reported previously that two such CTL clones recognize a product of the tyrosinase gene that is presented by HLA‐A2. Here we show that one of these CTL clones recognizes a peptide encoded by the first nine amino acids of the putative signal sequence of tyrosinase. The other CTL clone recognizes a different tyrosinase peptide corresponding to amino acids 368–376. Both peptides contain consensus motifs of HLA‐A2 binding peptides.
Identification of wild‐type and mutant p53 peptides binding to HLA‐A2 assessed by a peptide loading‐deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay
Authors: Stuber György , Leder Gerhard H. , Storkus Walter J. , Lotze Michael T. , Modrow Susanne , Székely László , Wolf Hans , Klein Eva , Kärre Klas , Klein George
Published: March 2007

Mutations of the p53 gene are the most frequently observed genetic changes in human cancers; often leading to an overexpression of the wild‐type (wt) p53 protein. Demonstrable T cell reactivity against tumor cells overexpressing wt or mutant p53‐derived peptides could support the application of such epitopes in cancer immunotherapies. As the binding of peptide to MHC class I molecules is a prerequisite for antigen‐specific T cell recognition, we evaluated the ability of wt and mutant p53 peptides to bind to HLA‐A2.1 using two independent flow cytometry‐based assay systems, the T2 major histocompatibility complex (MHC) class I peptide stabilization assay (stabilization assay) and the peptide‐induced MHC class I reconstitution assay (reconstitution assay). The twenty selected wt sequences each conformed to the previously reported HLA‐A2.1 peptide binding motif. Seven of the wt p53 and 2/13 mutant p53 peptides derived from the previously chosen wt peptides bound to HLA‐A2.1 in both the stabilization and the reconstitution assays. An additional six wt and six mutant p53 peptides, presumably exhibiting lower affinity for HLA‐A2.1, were identified only in the reconstitution assay. Those p53 peptides binding HLA‐A2.1 may provide useful immunogens for the generation of HLA‐A2.1‐restricted cytolytic T lymphocytes in vitro and in vivo.
Modulation of the transcripts for tumor necrosis factor‐α and its receptors in vivo
Authors: De Kossodo Sylvie , Critico Brigitte , Grau Georges E.
Published: March 2007

We investigated the effects of a single bacterial lipopolysaccharide (LPS) injection in vivo on the gene expression of tumor necrosis factor‐α (TNF) and its receptors: TNF receptor type I (TNF‐R 55 kDa or TNF‐R1) and TNF receptor type II (TNF‐R 75 kDa or TNF‐R2) in various tissues and white blood cells. While TNF mRNA rapidly accumulated in most tissues, TNF‐R1 and TNF‐R2 mRNA levels were found to be differentially regulated in lung, spleen, lymph nodes and white blood cells. In most cases, TNF‐R mRNA levels did not parallel TNF mRNA levels. These observations indicate that TNF‐R of both types are capable of modulating the host response to LPS, not only by shedding of their extracellular domains, but also by strict regulation of their gene expression.
A simple assay for detection of peptides promoting the assembly of HLA class I molecules
Authors: Connan Francine , Hlavac Frédéric , Hoebeke Johan , Guillet Jean‐GÉRard , Choppin Jeannine
Published: March 2007

Synthetic peptides derived from influenza virus and human immunodeficiency virus were tested for their ability to promote the assembly of HLA‐A2 and HLA‐B51 molecules in T2 cell lysates. Specific assembly was detected by an enzyme‐linked immunosorbent assay. The most significant HLA‐A2 assembly was obtained in the presence of peptides known to be targets for HLA‐A2‐restricted cytotoxic T lymphocytes (influenza matrix M.58–66 and HIV Pol 476–484). Three of a batch of Nef peptides corresponding to epitopic regions for cytotoxic T lymphocytes, caused significant assembly of HLA‐A2 (Nef 83–91, 137–145 and 144–153), but only at high concentrations (100 μM). As these peptides bound relatively weakly, it is unlikely that they are good candidates for HLA‐A2‐restricted CTL epitopes. Peptides matrix M.60–68, Nef 186–194, and Plasmodium falciparum sh. 77–85 produced the most significant assembly of HLA‐B51. These peptides have a dominant hydrophobic anchor residue (V, L. I) at position 9 that could occupy pocket “F”. Our results also suggest that another hydrophobic residue (V, L) at position 3 or 4 may anchor to hydrophobic pocket “d” of HLA‐B51. Proline at position 2 greatly increases HLA‐B51 anchoring.
B cells regulate expression of CD40 ligand on activated T cells by lowering the mRNA level and through the release of soluble CD40
Authors: van Kooten Cees , Gaillard Claude , Galizzi Jean‐Pierre , Hermann Patrice , Fossiez François , Banchereau Jacques , Blanchard Dominique
Published: March 2007

The expression of CD40 ligand (CD40L) on activated T cells (CD4+ T cell clone MT9) is diminished when the T cells are cultured in the presence of B cells. This effect, observed both with normal tonsil B cells and with the B cell line JY, was detected after 6 h and sustained at least until 18 h of co‐culture. Analysis of mRNA showed that CD40L mRNA levels were not modified after 6 h, but were significantly down‐regulated after 18 h of co‐culture with B cells. Although CD40L expression could not be detected by a CD40‐Fc chimera, the molecule was still expressed at the membrane as shown with a polyclonal antiserum against CD40L (anti‐TRAP). In addition, T cells activated in the presence of B cells were stained by a polyclonal antiserum against CD40, without the appearance of CD40 mRNA. These results indicated that a soluble form of CD40 (sCD40) bound to the expressed CD40L on T cells. The existence of sCD40 was confirmed by detection of sCD40 in B cell supernatants using a specific enzyme‐linked immunosorbent assay. Collectively, these data show that B cells can regulate the expression of CD40L on activated T cells at least by two different mechanisms.
Expression and function of the costimulatory molecule B7 on murine Langerhans cells: Evidence for an alternative CTLA‐4 ligand
Authors: Razi‐Wolf Ziba , Falo Louis D. , Reiser Hans
Published: March 2007

We have previously shown, through transfection experiments, that the murine B7 (mB7) molecule, a ligand for the CD28 and CTLA‐4 receptors, is a sufficient costimulatory signal for the antigen‐specific and major histocompatibility complex (MHC)‐restricted activation of murine CD4+ T lymphocytes. In addition to mB7, another ligand with affinity for CTLA‐4 has been described on spleen cells. Here we report our studies on the expression and function of these molecules on murine Langerhans cells (LC). Both anti‐mB7 monoclonal antibody (mAb) 16‐10A1 and human CTLA4Ig (hCTLA4Ig), a chimeric fusion protein consisting of the extracellular domain of human CLTA‐4 and the constant domain of human IgG1, detected antigens(s) on cultured but not freshly isolated LC. Preincubation of cultured LC with anti‐mB7 mAb did not significantly affect binding of hCTLA4Ig to these cells. This result demonstrate the existence of at least one other ligand for the CLTA‐4 receptor on cultured LC. Functional studies revealed that the costimulatory activity of LC was inhibited better by hCTLA4Ig than by the anti‐mB7 mAb. This differential effect was seen in the case of both alloreactive and antigen‐specific, syngeneic T cell responses. These findings suggest that the non‐mB7‐ligand for CTLA‐4 is functional and participates in the induction of immune responses by LC. Importantly, even synergistic combinations of anti‐mB7 mAb and hCTLA4Ig did not inhibit completely the activity of LC. These findings therefore raise the possibility that LC express other costimulatory ligands besides mB7 and related family members.
CD5 is associated with the human B cell antigen receptor complex
Authors: Lankester Arjan C. , van Schijndel Gijs M. W. , Cordell Jacqueline L. , van Noesel Carel J. M. , van Lier René A. W.
Published: March 2007

On human B cells the antigen receptor complex is composed of the membrane form of the immunoglobulin molecule and the non‐covalently associated Igα/β heterodimer. A small subpopulation of normal B cells and chronic lymphocytic leukemia B cells express (analogous to T cells) the transmembrane molecule CD5, a counterstructure of B cell‐specific CD72. Numbers of CD5+ B cells are increased in several physiological and pathological conditions. Moreover, CD5+ B cells are being held responsible for the production of autoreactive antibodies and seem to have signaling characteristics distinct from conventional B cells. On T cells, CD5 associates with the T cell receptor CD3 complex and ligation of CD5 leads to the generation of co‐stimulatory signals, that act on T cell activation. We here demonstrate that CD5 is associated with the B cell receptor (BCR) complex and serves as substrate for BCR‐induced tyrosine kinase activity. Hence, CD5+ B cells have a unique potential to modulate BCR signals.
A second promoter and enhancer element within the immunoglobulin heavy chain locus
Authors: Kottmann Andreas H. , Zevnik Branko , Welte Markus , Nielsen Peter J. , Köhler Georges
Published: March 2007

The joining of immunoglobulin gene segments during B cell development consists of a tightly regulated series of rearrangement steps. A variety of experiments have suggested that transcription is involved in activating the locus as substrate for the V(D)J recombinase. Here, we have characterized a region located immediately upstream of the most J‐proximal D element (DQ52), which contains both promoter and enhancer activities preferentially active in precursors of B cells. Interestingly, this DQ52 regulatory element is inevitably deleted in fully rearranged H chain genes. We propose that it is involved in the early activation and rearrangement events at the IgH locus.
Activation of CD4+ T cells by delivery of the B7 costimulatory signal on bystander antigen‐presenting cells (trans‐costimulation)
Authors: Ding Linna , Shevach Ethan M.
Published: March 2007

Increasing evidence in both murine and human systems suggests that the interaction of the T cell surface antigens CD28/CTLA4 with their ligand B7 on the antigen‐presenting cells (APC) is the critical costimulatory pathway involved in the induction of maximal T cell activation and the prevention of induction of anergy. It has also been demonstrated that efficient induction of clonal expansion of normal CD4+ T cells requires the delivery of the T cell receptor (TCR) ligand and costimulation by the same APC. We demonstrate here that normal murine CD4+ T cells can be efficiently activated by soluble anti‐CD3 cross‐linked by fixed macrophages and by a costimulatory signal delivered by a bystander APC, B7‐transfected L cells. The major factor which determined the ability of an APC to provide costimulation in “trans” was the level of cell surface B7 expression. The requirement for B7 costimulation appears to be at initial stage of TCR engagement since optimal T cell activation was only observed when TCR triggering and B7 costimulatory activity were delivered at same time by different APC. Induction of maximal proliferation of both naive CD45RBhi and memory CD45RBlo CD4+ T cells was B7 dependent and both populations of cells responded equally well to the B7 costimulation delivered in “trans”. Furthermore, trans‐costimulation provided by B7 transfected L cells efficiently prevented the induction of anergy in normal murine CD4+ T cells induced by anti‐CD3 cross‐linked by fixed‐resting macrophages. Addition of exogenous interleukin‐2 (IL‐2) and IL‐7 to the primary culture in the absence of B7‐transfected L cells or addition of IL‐2 to the culture containing the B7 transfectant and CTLA4Ig completely prevented the induction of hyporesponsiveness. These findings raise the possibility that in certain pathological states, CD4+ T cells in vivo may be activated by costimulation delivered by bystander APC.
B precursor acute lymphoblastic leukemia third complementarity‐determining regions predominantly represent an unbiased recombination repertoire: Leukemic transformation frequently occurs in fetal life
Authors: Steenbergen Eric J. , Verhagen Onno J. H. M. , van Leeuwen Eleonore F. , Behrendt Henk , Merle Pauline A. , Wester Mieke R. , Von Dem Borne Albert E. G. Kr. , van der Schoot C. Ellen
Published: March 2007

To determine whether the ALL (acute lymphoblastic leukemia) CDR3 (third complementarity‐determining region) repertoire represents the recombination repertoire, or shows evidence of selectional processes inherent to normal B cell differentiation or malignant transformation, we analyzed 68 ALL CDR3 regions and included 127 previously published sequences in the analyses. We found no evidence of selection prior to malignant transformation as recombination was random with 1/3 “in frame” and 2/3 “out of frame” joinings and usage of all three D reading frames was observed. D and JH gene segments were predominantly umutated which allowed a detailed analysis of gene usage and rearrangement characteristics. JH4 and JH6 usage (both 32.2%) was significantly different (p = 0.005) from that observed in peripheral B lymphocytes. D gene family usage roughly represented D gene family size with the exception of the DXP and DA/K family which were over‐ and underrepresented (p = 0.05), respectively. D‐D fusions were found in 26.2% of CDR3 regions. If less stringent criteria were applied DIR homology was found in 40/65 sequences, suggesting the frequent involvement of DIR gene segments in human CDR3 formation. The rearranged D genes were evenly distributed over the D locus, suggesting that D recombination is a predominantly random process, independent of physical location at the locus. Also, there was no correlation between JH gene usge and physical location of the rearranged D gene segment, which excludes a major contribution of the DJH replacement recombination mechanism. In 36.1% of CDR3 regions N‐nucleotides at the DJH junction were absent. This frequency is higher than observed for peripheral B lymphocytes. It is suggested that for a number of ALL the initial transformational event took place in early fetal life. We conclude that ALL CDR3 sequences show no evidence of selection prior to malignant transformation, nor of extensive changes subsequent to malignant transformation.
Interleukin‐12 but not interferon‐γ production correlates with induction of T helper type‐1 phenotype in murine candidiasis
Authors: Romani Luigina , Mencacci Antonella , Tonnetti Laura , Spaccapelo Roberta , Cenci Elio , Wolf Stanley , Puccetti Paolo , Bistoni Francesco
Published: March 2007

By means of polymerase chain reaction‐assisted mRNA amplification, we have monitored message levels of interleukin (IL)‐12 in splenic macrophages and of interferon‐γ (IFN‐γ), IL‐4, and IL‐10 in CD4+ and CD8+ T cells using Candida albicans/host combinations that result either in a T helper type‐1 (Th1)‐associated self‐limiting infection (“healer mice”) or in a Th2‐associated progressive disease (“nonhealer mice”). The timing and pattern of message detection did not differ qualitatively by the expression of IFN‐γ or IL‐10 mRNA in CD4+ and CD8+ cells from healer (i.e. PCA‐2 into CD2F1) vs. nonhealer (i.e. CA‐6 into CD2F1 or PCA‐2 into DBA/2) mice. In contrast, IL‐4 mRNA was uniquely expressed by CD4+ cells from nonhealer animals. IL‐12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen‐driven cytokine production by CD4+ and CD8+ cells was assessed in vitro, while IFN‐γ‐producing cells were enumerated in CD4− CD8− cell fractions. Overall, our results showed that (i) antigen‐specific secretion of IFN‐γ protein in vitro by CD4+ cells occurred only in healing infection; (ii) IL‐4‐ and IL‐10‐producing CD4+ cells would expand in nonhealer mice in the face of high levels of circulating IFN‐γ, likely released by CD4− CD8− lymphocytes; (iii) a finely regulated IFN‐γ production correlated in the healer mice with IL‐12 mRNA detection, and IL‐12 was required in vitro for yeast‐induced development of IFN‐γ‐producing CD4+ cells. Although the mutually exclusive production of IL‐4/IL‐10 and IFN‐γ by early CD4+ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL‐12 rather than IFN‐γ production may be an indicator of Th1 differentiation.
Fas‐based lymphocyte‐mediated cytotoxicity against syngeneic activated lymphocytes: A regulatory pathway?
Authors: Vignaux Françoise , Golstein Pierre
Published: March 2007

To investigate the possibility that Fas‐based immune regulation and Fas‐based T cell‐mediated cytotoxicity (F‐CMC) are causally related, we explored the latter in activated lymphocyte populations. These were shown to contain effector cells exerting cytotoxicity via an F‐CMC mechanism which could be differentially triggered by PMA and ionomycin. F‐CMC operated in trans, requiring an lpr (Fas) product on target cells and a gld product on effector cells. Activated lymphocyte populations were also shown to contain F‐CMC target cells. Activated lymphocyte populations thus contained both effector and target F‐CMC cells, which could lyse each other. Fas‐based cytotoxicity can thus lead to the lysis of syngeneic activated lymphocytes, consistent with the possibility of its participation in the down‐regulation of immune responses, and more generally offering a model of socially controlled, direct membrane‐mediated cell death.
CD3 components at the surface of pro‐T cells can mediate pre‐T cell development in vivo
Authors: Jacobs Heinz , Vandeputte Dmitri , Tolkamp Louis , De Vries Evert , Borst Jannie , Berns Anton
Published: March 2007

Developmentally arrested pro‐T cells (CD4−8−, IL‐2R+, HSA++) of RAG‐1‐deficient mice appear to express low levels of CD3 molecules in the absence of T cell receptor (TcR) chains at their surface, while developmentally arrested pre‐T cells of TcRα‐deficient mice express low levels of a disulfide‐linked TcRβ chain in association with CD3 molecules.

Cross‐linking of the CD3 modules on pro‐T cells of RAG‐1−/‐ mice in vivo, with either of two different CD3′‐specific monoclonal antibodies, induces differentiation of these pro‐T cells into pre‐T cells (CD4+8+, IL‐2R−, HSA+), concomitant with a rapid expansion of the thymic T cell compartment, up to 175‐fold within 12 days. The same effects can be produced by introduction of a mutant TcRβ transgene lacking most of the variable domain (ΔV‐TcRβ) into the RAG‐1−/‐ background.

These experiments suggest that cross‐linking of the CD3 modules on pro‐T cells mimics the signaling function expected of the pre‐TcR complex, which is found at the surface of pre‐T cells prior to functional TcRa gene rearrangement. The variable domain of the TcRβ chain is apparently not essential for inducing these aspects of T cell development.
Different requirements of ICAM‐1/LFA‐1 adhesion in allorecognition and self‐restricted antigen recognition by class II‐specific T cell clones
Authors: Jaraquemada Dolores , Martí Mercè , Martin Roland , Wagner Annette , MacFarland Henry F. , Rosen‐Bronson Sandra
Published: March 2007

We have analyzed the influence of non‐antigen‐specific interactions between ICAM‐1 and LFA‐1 in target recognition by allospecific and antigen‐specific Tcells at the clonal level, using human and mouse fibroblasts transfected with HLA‐DR1 or DR2 with or without co‐expression of ICAM‐1, as antigen‐presenting cells. The results show a great heterogeneity in the requirements for ICAM‐1/LFA‐1 interactions for antigen‐specific and alloreactive T cell responses and this requirement may depend on the avidity of any particular interaction. The data also show that for most alloreactive clones, ICAM‐1/LFA‐1 adhesion is not sufficient to facilitate efficient T cell recognition of its target molecule. HLA class II recognition by a large proportion of the DR1‐ and DR2‐specific alloreactive clones studied was different for class II molecules expressed on murine or human fibroblasts compared to human lymphoid cells, and was independent of ICAM‐1 expression on the stimulator cells. The inability of some T cell clones to recognize HLA‐class II expressed on non‐lymphoid cells suggests the absence of specific epitopes and could be due to the lack of the relevant peptides, either because they are derived from species‐specific proteins or to differences in processing of endogenous antigen in the transfected stimulator cells.
Distinct involvement of CD45 in antigen receptor signalling in CD4+ and CD8+ primary T cells
Authors: Maroun Christiane R. , Julius Michael
Published: March 2007

We demonstrate that pretreatment of primary CD4+, but not CD8+ T cells with anti‐CD45 inhibits activation signals induced through the T cell receptor for antigen (TCRαβ). Specifically, anti‐TCRαβ‐mediated tyrosine phosphorylation of phospholipase C‐γ1 is inhibited, and this in turn correlates with the inhibition of subsequent Ca2+ mobilization and DNA synthesis. In marked contrast, none of these activation parameters are affected by anti‐CD45 in CD8+ T cells. Perturbation of TCRαβ signalling in CD4+ cells is observed in conditions which do not detectably affect the level of CD45 expression, or its membrane distribution. Further, changes in the intrinsic phosphatase activity of CD45 are not detectable. While anti‐CD45 ablates TCRαβ signalling, anti‐CD3ϵ‐mediated activation is unaffected. This suggests that elements of the antigen receptor complex can be functionally uncoupled, and indicates that the requirements for CD45 in signalling through these two elements are different. The results demonstrate that the involvement of CD45 in coupling TCRαβ to second messenger‐generating pathways is under distinct physical and/or functional constraints in primary CD4+ and CD8+ T cells.
Plastic‐immobilized anti‐μ or anti‐δ antibodies induce apoptosis in mature murine B lymphocytes
Authors: Parry Sarah L. , Holman Mary J. , Hasbold Jhagvaral , Klaus Gerry G. B.
Published: March 2007

It is widely accepted that extensive cross‐linking of surface immunoglobulin (sIg) receptors on mature B cells promotes their activation and progression through the cell cycle. A commonly employed method to maximize receptor cross‐linking via anti‐receptor antibodies is to immobilize them on tissue culture plastic. We show here that immobilizing monoclonal anti‐μ or anti‐δ antibodies, which are mitogenic in solution, on plastic abrogates their capacity to induce DNA synthesis in mature murine B cells, even in the presence of interleukin‐4 (IL‐4). The cells do become abortively activated, as evidenced by up‐regulation of major histocompatibility complex class II antigen levels, but subsequently virtually all of them die, manifesting DNA fragmentation characteristic of apoptosis. The induction of apoptosis is abrogated by the inclusion of either IL‐4 or anti‐CD40 antibodies in the cultures, with the two stimuli acting in concert. We believe that the system represents a polyclonal model of clonal deletion tolerance in mature B cells, such as may be induced under physiological conditions by antigens with repeating epitopes.
A fungal metabolite which inhibits the interaction of CD4 with major histocompatibility complex‐encoded class II molecules
Authors: Gammon Guy , Chandler Glenda , Depledge Paul , Elcock Catherine , Wrigley Stephen , Moore John , Cammarota Giancarlo , Sinigaglia Francesco , Moore Michael
Published: March 2007

CD4, a cell‐surface glycoprotein expressed on a subpopulation of T cells, is the receptor for class II molecules of the major histocompatibility complex (MHC II) and a receptor for the envelope glycoprotein (gp 120) of human immunodeficiency virus‐1 (HIV‐1). Screening of microbial metabolites for CD4‐binding activity using an enzyme‐linked immunosorbent assay based on the binding of the CD4‐specific monoclonal antibody (mAb), anti‐Leu3a, identified a family of compounds comprising several novel polyketides. The parent compound (411F, Vinaxanthone) is a C28 molecule probably arising from a dimerization of two C14 polyketide units. It strongly inhibited the interaction of anti‐Leu 3a and that of several other D1/D2 epitope‐specific mAb with CD4, but only weakly inhibited the binding of HIV‐1 gp120. Binding of a representative MHC class II molecule, HLA‐DRB*0401, was also inhibited by 411F with a comparable inhibitory concentration (IC50 = 1 μM). In functional assays 411F inhibited antigen‐induced CD4‐dependent T cell proliferative responses of peripheral blood mononuclear cells. At the clonal level 411F exhibited selectivity in that the compound inhibited peptide‐induced CD4+ T cell proliferative responses but not alloantigen‐induced CD8+ T cell proliferation. It is hypothesized that 411F, a polyanionic compound in aqueous solution at neutral pH, inhibits CD4‐dependent functions by binding over a broad area of the positively charged amino‐terminal D1 and D2 domains implicated in the interaction with MHC II molecules. 411F has the potential for development as an immunosuppressive agent with a novel mechanism of action.
Small CD4+8+TCRlow thymocytes contain precursors of mature T cells
Authors: Swat Wojciech , Boehmer Harald Von , Kisielow Pawel
Published: March 2007

To evaluate directly the developmental potential of cortical CD4+8+ thymocytes, highly purified populations of small, nondividing CD4+8+TCRlow and large, dividing CD4+8+TCRhigh thymocytes from H‐2d mice expressing a transgenic T cell receptor restricted by H‐2Db (major histocompatibility complex class I) molecules were transferred into the thymus of normal, nonirradiated H‐2b recipient mice. The results show that both populations generate CD4−8+ thymocytes under these conditions, thus providing conclusive evidence that small cortical thymocytes do not represent a “dead end” but an important intermediate stage in T cell development.
Orally administered myelin basic protein in neonates primes for immune responses and enhances experimental autoimmune encephalomyelitis in adult animals
Authors: Miller Ariel , Lider Ofer , Abramsky Oded , Weiner Howard L.
Published: March 2007

Antigen‐driven tolerance is an effective method for suppression of autoimmune diseases. Adult animals can be tolerized against the induction of experimental autoimmune encephalomyelitis (EAE) by both oral and parenteral administration of myelin basic protein (MBP). We have found that in contrast to previous studies of neonatal tolerance in which parenterally administered autoantigens induced tolerance, the oral administration of MBP in neonatal rats did not result in tolerization to MBP, but instead, primed for immunologic responses. Proliferative responses to MBP and its encephalitogenic epitope were present in animals fed with MBP as neonates and co‐culture of encephalitogenic T cells with cells from neonatal rats fed with MBP were associated with enhanced MBP responses rather than the suppression observed with cells from adult rats fed with MBP. Furthermore, neonates fed with MBP and immunized 6–8 weeks later with MBP in adjuvant to induce EAE revealed enhancement of disease severity, and were not protected from a second attack upon active reinduction of EAE. Subcutaneous injection of soluble MBP into neonates had no effect on EAE induction as adults, whereas intraperitoneal injection of MBP in neonates was associated with marked suppression of disease in adults. Suppression of EAE began to appear in animals fed with MBP at 4 weeks of age, and was similar to oral tolerance in adult animals when animals were fed at 6 weeks of age. These results suggest that immaturity of the immunoregulatory network associated with oral tolerance and sensitization to autoantigens via the gut in the neonatal period may contribute to the pathogenesis of autoimmune diseases.