bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphatidylinositol 4,5‐bisphosphate hydrolysis accompanies T cell receptor‐induced apoptosis of murine thymocytes within the thymus
Authors: Conroy Louise A. , Jenkinson Eric J. , Owen John J. T. , Michell Robert H.
Published: March 2007

Regulation of the development of thymocytes into mature T cells within the thymus is now known to involve antigen‐induced deletion, by apoptosis, of potentially autoreactive thymocytes, and it can be mimicked either by stimulating the T cell receptor (TcR) complex by monoclonal antibody (mAb) or by ionophore‐induced elevation of cytosolic [Ca2+]. To identify signaling pathways employed by the TcR complex of immature thymocytes, we examined the effects of anti‐CD3 and anti‐TcRß constant (c) region mAb, staphylococcal enterotoxin B (SEB) and pharmacological agents on the generation of inositol phosphates through hydrolysis of phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] both in cultured fetal mouse thymic lobes and in the CD4+CD8+ immature thymocyte cell line, TM10G. Stimulation of the TcR complex with anti‐CD3 mAb provoked an accumulation of inositol phosphates diagnostic of the occurrence of receptor‐stimulated phosphoinositidase C (PLC) activation. Anti‐TcRCß mAb and SEB provoked smaller but similar responses. The PLC activation evoked by anti‐CD3 mAb was suppressed by inhibitors of receptor tyrosine kinases and was unmodified by protein kinase C activation or elevation of cytosolic [Ca2+]. It thus appears that apoptosis triggered by TcR stimulation is associated with PLC activation by a receptor‐regulated tyrosine kinase. Treatment of thymic lobes or TM10G cells with fluoroaluminate provoked apoptosis of a wider range of thymocyte subtypes and such stimulation also provoked an accumulation of inositol phosphates. The responses to fluoroaluminate were not prevented by inhibitors of tyrosine kinases, suggesting that unidentified GTP‐binding proteins which couple to PLC activation may also be capable of initiating apoptosis by a route independent of the TcR. These results, when considered alongside previous studies of mature T cells, indicate that stimulation of immature thymocytes or of mature T cells through their TcR complex activates the PLC‐catalyzed PtdIns(4,5)P2 hydrolysis signaling pathway, and thus that this signaling pathway may be implicated both in provoking apoptosis in immature T cells and in initiating proliferation in mature T cells.
Germinal‐center cell proliferation in response to T‐independent antigens: A stathmokinetic, morphometric and immunohistochemical study in vivo
Authors: Goodlad John R. , Macartney James C.
Published: March 2007

Although the nature of the germinal center reaction during responses to T‐dependent antigens has been well documented, much less is known regarding the relationship between germinal centers and T‐independent antigens. In this study, germinal‐center cell proliferation was determined at specific time points in spleens of C3H/HeN mice following immunization with either the type‐1, T‐independent antigen dinitrophenol‐lipopolysaccharide (DNP‐LPS), or the type‐2, T‐independent antigen DNP‐Ficoll. A stathmokinetic technique was employed to assess proliferation in terms of germinal center cell birth rate and morphometry was used to measure actual growth and regression of the germinal center cell population. An estimate of the absolute rate of germinal‐center (GC) cell proliferation was derived from these two values. In addition, immunohisto‐chemistry was performed to correlate changes in GC cell proliferation with the presence or absence of antigen within GC. Following immunization with both antigens, there was an initial reduction of proliferation within pre‐existing germinal centers which manifested as either GC dissociation (DNP‐LPS) or a suppression of birth rates (DNP‐Ficoll). This was followed by a period of increased GC cell proliferation in animals immunized with DNP‐LPS, but not in those exposed to DNP‐Ficoll. GC cell proliferation was then measured in mice treated with cyclosporin A from 1 day before to 2 days after immunization with DNP‐LPS. In these animals, the expected increase in GC cell birth rates did not take place. Immunohistochemistry showed that DNP‐Ficoll and DNP‐LPS were present in GC from 1 day after immunization until the end of the experiment on day 7. Treatment with cyclosporin A did not affect the deposition of DNP‐LPS in GC. These results show that only some T‐independent antigens are able to stimulate GC cell proliferation, and we propose that this is related to their ability to recruit precursors of GC B cells into the GC reaction. In addition, the results indicate that GC proliferation seen in response to a so‐called T‐independent antigen is at least partly driven by T cell‐derived cytokines.
Peripheral deletion of autoreactive CD8+ T cells in transgenic mice expressing H‐2Kb in the liver
Authors: Bertolino Patrick , Heath William R. , Hardy Charles L. , Morahan Grant , Miller Jacques F. A. P.
Published: March 2007

The response of T cells specific for liver antigens was examined in transgenic mice expressing the allogeneic major histocompatibility complex class I molecule H‐2Kb (Kb) under the control of the sheep metallothionein promoter (Met‐Kb mice). To follow the fate of Kb‐specific T cells, and to prevent any aberrant thymic expression of the Kb transgene, the mice were thymectomized, lethally irradiated, protected with bone marrow cells from transgenic mice expressing in their T cells a Kb‐specific T cell receptor identifiable by a clonotypic antibody, and given syngeneic non‐transgenic thymus grafts. Although Kb‐specific CD8+ T cells were produced in the thymus grafts of these manipulated Met‐Kb mice, only small numbers of such cells could be detected in the spleen and lymph nodes. The livers, however, showed signs of damage and were heavily infiltrated by actively dividing CD8+ T cells. We provide strong evidence that the hepatocytes, not generally regarded as antigen‐presenting cells, activated the Kb‐specific CD8+ T cells and that these disappeared after a vigorous autoimmune response that resulted in deletion.
IgD expression on B cells is more efficient than IgM but both receptors are functionally equivalent in up‐regulation CD80/CD86 co‐stimulatory molecules
Authors: Brink Robert , Goodnow Christopher C. , Basten Antony
Published: March 2007

The expression and function of IgM and IgD antigen receptors were studied in a series of anti‐hen egg lysozyme (HEL) immunoglobulin (Ig)‐transgenic mice expressing either IgM alone, IgD alone, or both IgM and IgD. B cell surface expression of IgD was found to be more efficient than that of IgM. Thus antigen receptor density on IgD+, IgM− B cells was twofold higher than on IgM+, IgD− B cells despite the presence of sevenfold lower levels of membrane heavy chain mRNA, and coexpression of IgD with IgM led to almost complete inhibition of surface IgM. In addition, less extensive down‐regulation of IgD occurred following exposure to antigen in vitro. When regulation of CD80/CD86 co‐stimulatory molecules by surface Ig was examined, up‐regulation of the former was initiated at lower antigen concentrations on IgM−, IgD+ compared to IgM+, IgD− B cells. On correcting for antigen receptor density, however, induction of CD80/CD86 by IgM and IgD was comparable. Taken together, these results reinforced the functional similarity of IgM and IgD antigen receptors while at the same time revealing differences in expression which may explain their simultaneous presence on mature B cells.
Mechanism of enhanced antigen presentation by B cells activated with anti‐μ plus interferon‐γ: Role of B7‐2 in the activation of naive and memory CD4+ T cells
Authors: Morokata Tatsuaki , Kato Takuma , Igarashi Osamu , Nariuchi Hideo
Published: March 2007

B cells activated with anti‐γ antibody plus interferon (IFN)‐γ exerted strong antigen presentation activity for T cell proliferation. The enhanced antigen presentation function was shown to be due to the increase in B7‐2 expression. When B cells were stimulated with anti‐μ, expression of MHC major histocompatibility complex class II, heat‐stable antigen (HSA), ICAM‐1 and B7‐2 was increased. The presence of IFN‐γ further augmented the expression of B7‐2 on anti‐μ‐stimulated B cells. B7‐1 was not expressed on B cells under these conditions. The participation of B7‐2 in the elicitation of the proliferative response of T cells was confirmed by the inclusion of anti‐B7‐2 antibody in cultures. The enhanced expression of either HSA or ICAM‐1 was shown not to play a major role in the increased B cell antigen presentation capacity. The major T cell population responding to this activated B cell antigen presentation was shown to be CD44low naive CD4+ T cells, whereas CD45RBlow memory CD4+ T cells responded only weakly. The difference in proliferative responses between naive and memory CD4+ T cells was explained by the different efficiency in IL‐2 production of these cell populations in response to antigen presentation by B cells activated by anti‐μ plus IFN‐γ. These results suggest that IFN‐γ plays an important role in recruitment of naive T cells for an immune response.
Self and non‐self peptides treat autoimmune encephalomyelitis: T cell anergy or competition for major histocompatibility complex class II binding?
Authors: Gautam Anand M.
Published: March 2007

In susceptible strains of mice, myelin basic protein (MBP) peptide Ac1‐11 induces experimental autoimmune encephalomyelitis (EAE) providing a useful model for human multiple sclerosis. Ac1‐11 binds major histocompatibility complex (MHC) class II molecules AαuAβu. Here, we show that the Ac1‐11 peptide, when administered intraperitoneally in incomplete Freund's adjuvant (IFA) emulsion, can effectively treat Ac1‐11‐induced EAE in mice. Treatment with Ac1‐11/IFA 9 days after initial immunization with Ac1‐11 in complete Freund's adjuvant (CFA) results in a loss of T cell proliferation to MBP Ac1‐11. This lack of T cell proliferation is not due to T cell anergy and is not specific. A similar lack of T cell proliferation and inhibition of EAE is observed when an ovalbumin peptide OVA323–339 or a sperm whale myoglobin peptide SWM110‐121 are used to treat mice immunized with Ac1‐11. Interestingly, we show that previously unresponsive lymph node cells from treated mice respond normally if Ac1‐11 is presented by fresh antigen‐presenting cells taken from normal mice. These results argue that the lack of T cell proliferation and inhibition of EAE is not due to specific T cell anergy as suggested by others. Instead this appears to be due to blocking of MHC class II molecules AαuAβu by the treating peptides.
Activation of human peripheral blood dendritic cells induces the CD86 co‐stimulatory molecule
Authors: McLellan Alexander D. , Starling Gary C. , Williams Lisa A. , Hock Barry D. , Hart Derek N. J.
Published: March 2007

Maximal T lymphocyte responses require presentation of antigen by major histocompatibility complex molecules and delivery of one or more co‐stimulatory signals. Interaction of the CD28 molecule on T lymphocytes with its ligands on antigen‐presenting cells (APC) initiates a critical co‐stimulatory pathway inducing T lymphocyte proliferation and cytokine secretion. Dendritic cells (DC) are potent APC for a primary T lymphocyte response and potential CD28/CTLA‐4 ligands on DC are, therefore, of particular functional relevance. In these experiments, the expression and function of the CD28/CTLA‐4 ligands B7.1 (CD80) and B7.2 (CD86) were examined on human blood DC. Resting DC populations directly isolated by immunodepletion of lineage marker‐positive cells lacked cell membrane expression of CD80 and expressed little or no CD86, although CD86, but not CD80 mRNA was detected by reverse transcription‐polymerase chain reaction analysis. In contrast, low‐density DC isolated after culture in vitro strongly expressed CD86 surface protein, but expressed limited or no CD80, although mRNA for both molecules were detected. Short‐term culture of directly isolated DC up‐regulated both CD80 and CD86 expression. Analysis of the kinetics of CD28/CTLA‐4 ligand induction showed that surface CD86 was present within 8 h, whereas CD80 antigen was first detected after 24 h of culture. The functional importance of CD28/CTLA‐4 ligand up‐regulation on DC during T lymphocyte interactions was demonstrated by the ability of both CTLA‐41g and CD86 monoclonal antibodies (mAb), but not CD80 mAb, to block an allogeneic mixed lymphocyte reaction stimulated by DC populations initially negative for CD80 and CD86. These results demonstrate that CD86 is both the earliest and functionally the predominant co‐stimulatory CD28/CTLA‐4 ligand on DC.
Selective killing of T cells by immunotoxins directed at distinct Vβ epitopes of the T cell receptor
Authors: Rigaut Kathleen D. , Scharff Joshua E. , Neville David M.
Published: March 2007

The potency and specificity of anti‐T cell receptor (TcR)‐directed immunotoxins were studied in two T cell leukemia lines, HPB‐ALL and Jurkat, and in primary T cells. Immunoconjugates were synthesized using anti‐CD3ϵ or distinct anti‐Vβ antibodies cross‐linked to CRM9, a binding site‐mutant of diphtheria toxin. All TcR‐expressing cells display the CD3 complex on the plasma membrane. HPB‐ALL cells express the Vβ5 gene product in the β subunit of the TcR, while Jurkat cells express Vβ8. Vβ expression in primary T cells isolated from buffy coats is heterogeneous. Primary T cell populations expressing specific Vβ epitopes in the TcR were generated by plating CD3+ T cells on Vβ‐specific antibody‐coated flasks or by positive immunomagnetic selection. Immunotoxins directed against the invariant CD3ϵ epitope target and kill all T cells. Immunoconjugates targeted at distinct anti‐Vβ epitopes are specific for cells that express the corresponding gene product in the TcR. The results demonstrate the ability of antiTcR‐based immunotoxins selectively to kill T cells with defined Vβ epitopes. These reagents may be clinically useful in disorders mediated by autoreactive T cell populations exhibiting Vβ restriction and in the treatment of clonal TcR‐expressing lymphomas.
Recombinant CD30 ligand and CD40 ligand share common biological activities on Hodgkin and Reed‐Sternberg cells
Authors: Gruss Hans‐Jürgen , Ulrich Dawn , Braddy Steve , Armitage Richard J. , Dower Steven K.
Published: March 2007

The CD30 ligand (CD30L) and CD40L are members of the tumor necrosis factor (TNF) protein superfamily. CD30L and CD40L are mainly expressed as membrane‐bound proteins by activated T cells. CD30L and CD40L are costimulatory for T cell proliferation and activation. Further, CD40L is a critical signal for T cell‐dependent activation of B cells. Primary and cultured Hodgkin and Reed‐Sternberg (H‐RS) cells, the neoplastic component of Hodgkin's disease (HD), express high levels of the counterreceptors CD30 and CD40. We have found that both the recombinant CD30L and CD40L enhanced interleukin (IL)‐6, TNF and lymphotoxin (LT)‐α release from cultured H‐RS cells. In addition, CD40L, but not CD30L, induced IL‐8 secretion. CD30L and CD40L seem to share some redundant biological activities involved in the deregulated secretion of cytokines known to play a central role in the clinical presentation and pathology of HD. Further, CD30L enhanced surface expression of intercellular adhesion molecule‐1 (ICAM‐1/CD54) on cultured H‐RS cells, which is frequently overexpressed on primary H‐RS cells. CD30L‐ and CD40L‐enhanced CD54 surface expression is followed by elevated shedding of CD54, as shown by detection of elevated 82‐kDa soluble (s) CD54 levels in culture supernatants after stimulation with both ligands. CD30L and CD40L share common pleiotropic biological activities on CD30+/CD40+ H‐RS cells and are elements of the cytokine and cell contact‐dependent activation network typical for HD, a tumor of cytokine producing cells.
Analysis of interleukin receptor gene expression in mouse fetal liver by quantitative polymerase chain reaction
Authors: Jia Gui‐Quan , Gutierrez‐Ramos Jose C.
Published: March 2007

We describe a simplified and sensitive polymerase chain reaction (PCR)‐based method for the quantification of low‐abundance RNA for mouse cytokine receptor genes. Accurate quantification is achieved in a two‐step protocol which uses a synthetic RNA as an internal standard. The proper titration of the amount of mRNA molecules is followed by a kinetic analysis which ensures precise measurement. This quantitative PCR method provides a rapid and reliable way to quantify the amount of cytokine receptor mRNA in samples containing as few as 1000 molecules of RNA for a cytokine receptor target gene. We illustrate our approach by quantifying mRNA levels for two families of cytokine receptor genes in the fetal liver and bone marrow of the mouse. Our results reveal early and abundant expression of the genes encoding the signal transducing subunits interleukin‐2 receptor γ and gp130. Their expression seems to precede that of the genes encoding the specific subunits of these interleukin receptor systems.
T cell repertoire and clonal deletion of Mtv superantigen‐reactive T cells in mice lacking CD4 and CD8 molecules
Authors: Penninger Josef M. , Schilham Marco W. , Timms Emma , Wallace Valerie A. , Mak Tak W.
Published: March 2007

CD4−CD8− double‐negative T cells constitute a lymphocyte subpopulation within the thymus and peripheral lymphatic organs that express a unique T cell receptor (TCR) repertoire and do not undergo negative selection. To test whether these cells develop as a distinct lineage or due to altered selection in the absence of CD4 and CD8 expression, we analyzed the TCR repertoire in mice lacking both CD4 and CD8 accessory molecules after homologous recombination (CD40/0CD80/0). We show that mature T cells of CD40/0CD80/0 mice express an unbiased diverse TCR Vβ repertoire comparable to wild type mice. In addition, clonal deletion of mouse mammary tumor virus superantigen‐reactive T cells did occur in CD40/0CD80/0 mice. These data show that the intrinsic lack of CD4 and CD8 expression has no effect on the mature TCR repertoire and that clonal deletion of superantigen‐reactive cells is independent of CD4 and CD8 co‐receptors.
IgA production by peritoneal cavity B cells is IL‐6 independent: Implications for intestinal IgA response
Authors: Beagley Kenneth W. , Bao Shisan , Ramsay Alistair J. , Eldridge John H. , Husband Alan J.
Published: March 2007

We have shown previously both in vitro and in vivo that IL‐6 is an important factor for the development of IgA‐producing B cells. However, despite the lack of this cytokine in mice with targeted disruption of the interleukin (IL)‐6 gene (gene knockout mice), a substantial number of IgA‐producing plasma cells occur in their intestinal mucosa. The experiments reported here indicate that there is a population of IgA‐producing B cell precursors originating from the peritoneal cavity, distinguished from conventional Peyer's patch‐derived precursors by their expression of CD5, and that IgA secretion by these cells is IL‐6‐independent. Further, there is an increase in CD5 expression among brightly staining IgA‐producing cells obtained from the intestinal lamina propria of IL‐6 gene‐disrupted mice compared to normal controls. These data suggest an explanation for the persistence of IgA‐producing plasma cells in the intestinal mucosa of IL‐6‐depleted mice and indicate the importance of IL‐6 for development of conventional precursors of IgA‐producing B cells, but not those derived from the peritoneal cavity pool.
Interleukin‐1β converting enzyme in murine Langerhans cells and epidermal‐derived dendritic cell lines
Authors: Ariizumi Kiyoshi , Kitajima Toshiyuki , Bergstresser Paul R. , Takashima Akira
Published: March 2007

Interleukin (IL)‐1β plays an essential role in the induction of T cell‐mediated immune responses in skin. Langerhans cells (LC), which constitutively express IL‐1β mRNA, have been assumed to be the primary source of IL‐1β in murine epidermis. The purpose of this study was to determine whether LC express mRNA for the IL‐1β converting enzyme (ICE), a protease that is required for processing pro‐IL‐1β into an active form. Here, we report that both IL‐1β and ICE mRNA are expressed by the Ia+ population (i.e. LC) in murine epidermis. Moreover, murine epidermal‐derived DC lines (XS series) also express both IL‐1β and ICE mRNA, and they secrete relatively large amounts of IL‐1β following lipopolysaccharide (LPS) stimulation. Finally, LPS‐triggered IL‐1β secretion by XS cells is blocked almost completely by the ICE inhibitor acetyl‐Tyr‐Val‐Ala‐Asp‐CH2OC(O)‐[2,6‐(CF3)2]Ph. These results demonstrate that LC are the primary source of IL‐1β within the epidermis, and suggest that the proinflammatory role of IL‐1β may be regulated pharmacologically by ICE inhibitors in vivo.
TAP polymorphism does not influence transport of peptide variants in mice and humans
Authors: Obst Reinhard , Armandola Elena A. , Nijenhuis Marga , Momburg Frank , Hämmerling Günter J.
Published: March 2007

The major histocompatibility complex (MHC)‐encoded transporter associated with antigen processing (TAP) delivers cytosolic peptides to the lumen of the endoplasmic reticulum (ER) for presentation by MHC class I molecules. For the rat, it has been demonstrated that TAP polymorphism results in the selection of different sets of peptides, the nature of the C terminus being of particular importance. Here, we investigated whether TAP polymorphism in mice and humans has functional consequences for transport of peptide sets variable at the C‐terminal residues. Using cell lines of H‐2d, H‐2k, and H‐2dxk haplotype and a panel of human lymphoblastoid cell lines expressing eight different TAP alleles, we detected species‐specific transport patterns, but no significant influence of TAP polymorphism on peptide selection. In addition, peptides with different core sequences were translocated to the same extent by different TAP. These results suggest that a major contribution of human TAP polymorphism to disease progression and autoimmunity is not very likely.
Interleukin‐2 promoter activity in Epstein‐Barr virus‐transformed B lymphocytes is controlled by nuclear factor‐ϰB
Authors: Mouzaki Athanasia , Serfling Edgar , Zubler Rudolf H.
Published: March 2007

The regulation of interleukin (IL)‐2 gene expression has been investigated mainly in T lymphocytes, the predominant producers of IL‐2. However, B cells can also synthesize IL‐2. In the present study we analyzed the control of IL‐2 promoter activity in Epstein‐Barr virus (EBV)‐transformed B cell clones which are capable of secreting IL‐2 at a low level after stimulation with phorbol 12‐myristate 13‐acetate and the Ca2+ ionophore ionomycin. Transient transfections using reporter constructs with multiples of transcription factor binding sites from the IL‐2 promoter [distal nuclear factor (NF)‐AT, proximal NF‐AT, AP‐1/Octamer (UPS) or NF‐ϰB (TCEd) sites] were performed. In EBV‐transformed B clones, the ϰB site exerted the strongest inducible activity; the NF‐AT binding sites showed either no or only weak activity compared to Jurkat T cells. An IL‐2 promoter bearing a defective NF‐ϰB site was completely inactive in EBV‐B cells, white it still had activity in Jurkat T cells. In seven EBV‐B cell clones or lines differing in their capacity to secrete IL‐2, the activity of the IL‐2 promoter correlated well with the status of IL‐2 secretion. Similarly, a human immunodeficiency virus promoter, whose activity is controlled through ϰB factors, was found to be active in the IL‐2‐producing EBV‐B cells, but inactive in the non‐IL‐2‐producing cells. Electrophoretic mobility shift assays using protein extracts from EBV‐B cells and the IL‐2 NF‐ϰB probe revealed the constitutive generation of ϰB complexes in IL‐2‐secreting cells consisting mainly of heterodimeric p50/p65 complexes. A weaker ϰB complex formation and faster‐migrating complexes were detected in non‐IL‐2‐secreting cells. These results demonstrate that the IL‐2 NF‐ϰB site is indispensable for the activity of the IL‐2 promoter in EBV‐transformed B cells, whereas other transcription factors appear to be less important for IL‐2 expression in these cells.
Ligation of HLA class II molecules promotes sensitivity to CD95 (Fas antigen, APO‐1)‐mediated apoptosis
Authors: Yoshino Tadashi , Cao Liu , Nishiuchi Ritsuo , Matsuo Yoshinobu , Yamadori Ichiro , Kondo Eisaku , Teramoto Norihiro , Hayashi Kazuhiko , Takahashi Kiyoshi , Kamikawaji Nobuhiro , Akagi Tadaatsu
Published: March 2007

CD95 (Fas antigen/APO‐1) is up‐regulated in activated lymphocytes, and monoclonal antibody (mAb) to CD95 induces apoptosis. HLA class II molecules play a key role in antigen presentation, ligation of which induces signal transduction. We examined 18 lymphoid cell lines (15 B cell and 3 T cell lines) to investigate the effects of ligation of HLA class II molecules on CD95‐mediated apoptosis. All of the five immature B cell lines were sensitive to anti‐CD95 mAb, and ligation of HLA class II molecules promoted CD95‐mediated apoptosis. In seven B‐blastoid cell lines, two Burkitt lines were resistant to anti‐CD95 mAb in spite of high expression of CD95. In three of five non‐Burkitt B‐blastoid lines, CD95‐mediated apoptosis was augmented by treatment with anti‐HLA class II mAb, while the other two lines lacking CD95 were resistant to anti‐CD95 mAb. Three plasmacytic cell lines showed CD95‐mediated apoptosis, but enhancement by anti‐HLA class II mAb was slight in one cell line and was not observed in the other two lines. Out of three HLA class II antigen‐positive T cell lines, CD95‐mediated apoptosis was observed to some degree in one cell line but was not promoted by the treatment with anti‐HLA class II mAb, and the other two cell lines were resistant to anti‐CD95 mAb. Ligation of HLA class II molecules did not alter CD95 expression in the five cell lines examined, except Su‐DHL‐4 originated from a follicular lymphoma, which showed slight up‐regulation. Taken together, ligation of HLA class II molecules apparently promotes CD95‐mediated apoptosis in immature B cells and non‐Burkitt B blasts. These findings highlight the role of HLA class II molecules in CD95‐mediated apoptosis, which may facilitate rapid clearance of functionally useless cells from the immune system and might be involved in negative selection of B cells.
Schistosoma‐specific helper T cell clones from subjects resistant to infection by Schistosoma mansoni are Th0/2
Authors: Couissinier‐Paris Patricia , Dessein Alain J.
Published: March 2007

Although T helper cells play a critical role in human immunity against schistosomes, the properties of the T lymphocytes that govern resistance and pathogenesis in human schistosomiasis are still poorly defined. This work addresses the question as to whether human resistance to Schistosoma mansoni is associated with a particular T helper subset. Twenty‐eight CD3+, CD4+, CD8− parasite‐specific T cell clones were isolated from three adults with high degree of resistance to infection by S. mansoni. The lymphokine secretion profiles of these clones were determined and compared to those of 21 CD3+, CD4+, CD8− clones with unknown specificity, established from these same subjects in the same cloning experiment. Almost all parasite‐specific clones produced interleukin (IL)‐4 and interferon (IFN)‐γ in large amounts. However, they generally produced more IL‐4 than IFN‐γ; variations in IL‐4/IFN‐γ ratios were accounted for by differences in IFN‐γ production since IL‐4 levels were comparable for the clones from the three subjects. T cell clones of unknown specificity produced significantly less IL‐4 and more IFN‐γ than parasite‐specific T cell clones. Most clones produced IL‐2, and IL‐2 production did not differ between the two types of clones. Parasite‐specific T cell clones from the resistant subjects were compared to specific T cell clones from a sensitized adult from a nonendemic area: T cell clones from this latter subject were the highest IFN‐γ and the lowest IL‐4 producers, compared to those of resistant subjects. Thus, parasite‐specific T cell clones isolated from adults resistant to S. mansoni belong to the Th0 subset and produced more IL‐4 than IFN‐γ (Th0/2), whereas clones of a sensitized adult from a nonendemic area are also Th0, but produce more IFN‐γ than IL‐4 (Th0/1). These results support previous conclusions on the role of IgE in protection against schistosomes in humans, and may indicate that IFN‐γ is required for full protection.
Expression of FcγRIII defines distinct subpopulations of fetal liver B cell and myeloid precursors
Authors: Carlsson Leif , Candéias Serge , Staerz Uwe , Keller Gordon
Published: March 2007

We have isolated four distinct fetal liver (FL) populations based on the expression of AA4.1 and the low‐affinity Fcγ receptors type II and III (FcγRII/III), and characterized them with respect to B cell, T cell, and myeloid precursor content. Polymerase chain reaction analysis revealed that the prevalent FcγR isoform at this stage of FL development (day 12 of gestation) was FcγRIII. Two of the four populations, one which expressed AA4.1 but little if any FcγRII/III (AA4.1+), and one which expressed abundant levels of both markers (AA4.1+/ FcR+), contained B cell precursors that grew and differentiated to generate VHDJH‐rearranged B‐lineage cells on S‐17 stromal cells in the presence of IL‐7. When cultured on FLST2 stromal cells only the AA4.1+ cells generated VHDJH‐rearranged B‐lineage cells. T cell precursors as assayed by their ability to repopulate fetal thymi in organ culture were found only in the AA4.1+ fraction. In contrast to the lymphoid precursors, myeloid precursors able to generate colonies in methyl cellulose cultures were found in all four fractions including the one which expressed FcγRII/III but no AA4.1 (FcR+) and the one which expressed neither marker (AA4.1−/FcR−). The AA4.1+ population which contained both B cell and T cell precursors was enriched for precursors from many myeloid lineages including the most immature ones which generated multilineage colonies. In contrast, the AA4.1+/FcR+ population, which also contained B cell precursors, was almost devoid of myeloid precursors and the few that were detected were committed to the macrophage lineage. The population defined as FcR+ was also enriched for precursors; however, the majority of these were committed to the erythroid, the macrophage and the mast cell lineage. The fourth population which expressed neither marker (AA4.1−/FcR−) was enriched for relatively mature erythroid precursors which were not present in any of the other fractions. Together, these findings demonstrate that fractionation of FL cells on the basis of AA4.1 and FcγRII/III expression distinguishes subpopulations of B cell and myeloid precursors and suggests that the low‐affinity FcγRIII could play a role in the development of early hematopoietic cells at this stage of ontogeny.
Both allelic forms of the rat T cell differentiation marker RT6 display nicotinamide adenine dinucleotide (NAD)‐glycohydrolase activity, yet only RT6.2 is capable of automodification upon incubation with NAD
Authors: Haag Friedrich , Andresen Viola , Karsten Stefan , Koch‐Nolte Friedrich , Thiele Heinz‐Günter
Published: March 2007

The finding that recently cloned mono‐ADP‐ribosyltransferases show sequence similarity to the rat T cell differentiation marker RT6 has led us to investigate the enzymatic activity of this alloantigenic system. To search for ADP‐ribosylation of cell surface proteins, T cell populations from RT6.1‐ and RT6.2‐expressing rat strains, as well as RT6.1+ and RT6.2+T‐T hybridoma cell lines, were incubated with [32P]nicotinamide adenine dinucleotide (NAD). All RT6.2+, but no RT6.1+ or RT6− cells, show incorporation of radioactivity into a single protein which could be identified as RT6.2 by immunoprecipitation with monoclonal antibodies. This automodification of RT6.2 is covalent, requires intact NAD as substrate, and displays characteristics typical for linkage of ADP‐ribose to arginine. The alloantigens RT6.1 and RT6.2 differ in ten amino acids, RT6.2 having two arginine residues not present in RT6.1. Both alloantigens were found to display potent NAD‐glycohydrolase activity.
Transgenic mice expressing high levels of soluble TNF‐R1 fusion protein are protected from lethal septic shock and cerebral malaria, and are highly sensitive to Listeria monocytogenes and Leishmania major infections
Authors: Garcia Iréne , Miyazaki Yoshitaka , Araki Kimi , Araki Masatake , Lucas Ralf , Grau Georges E. , Milon Geneviéve , Belkaid Yasmine , Montixi Christine , Lesslauer Werner , Vassalli Pierre
Published: March 2007

Mice bearing a transgene coding for a soluble tumor necrosis factor receptor type 1 (TNFR1)‐FeIgG3 fusion protein and placed under the control of the alpha‐1‐antitrypsin gene promoter were generated. Depending on the mouse line, blood levels of the protein ranged from 25 ng/ml to over 100 μg/ml; this level of expression was most often transmitted to the transgene‐bearing progeny as a relatively stable feature. High‐expressor mice were completely resistant to lipopolysaccharide‐induced shock and lethality, including after D‐galactosamine sensitization, and mice expressing about 1 μg of the fusion protein/ml were partially (60%) protected. In contrast, mice expressing less than 0.1 μg of the protein/ml were more sensitive than controls with respect to incidence and time of death, even though the biological activity of serum tumor necrosis factor (TNF) was partially neutralized. High‐expressor mice of the adequate genetic background were markedly, although not completely, protected from death by cerebral malaria after injection with Plasmodium berghei. They were highly susceptible to Listeria monocytogenes, dying from bacterial dissemination after sublethal infection, and to Leishmania major, displaying severe, non‐healing lesions after local infection. Under the same conditions, mice expressing about 1 μg protein/ml were only partially sensitive to these last agents, compared to nontransgenic littermate mice which were fully resistant. These transgenic mice represent a model of permanent, complete or partial, impairment of TNF use, which compares favorably, for ease of breeding and for the range of effects, to mice bearing gene disruptions.
Apoptotic death of lymphocytes in murine acquired immunodeficiency syndrome: Involvement of Fas‐Fas ligand interaction
Authors: Kanagawa Osami , Vaupel Barbara A. , Korsmeyer Stanley J. , Russell John H.
Published: March 2007

Murine acquired immunodeficiency syndrome (MAIDS) is caused by a defective murine leukemia virus. The disease is characterized by abnormal lymphoproliferation, impaired T and B cell function and aberrant regulation of cytokines. Both T and B lymphocytes show activated phenotypes, but undergo apoptotic death with characteristic DNA fragmentation. These results indicate the presence of a continuous activation death pathway of the lymphocytes in MAIDS. Overexpression of the bcl‐2 transgene in lymphocytes showed no effect on the apoptotic cell death or on the development of the disease. In contrast, mice carrying mutations in either Fas or Fas ligand exhibited accelerated progression of the disease upon infection with MAIDS virus. These results suggest the involvement of Fas‐Fas ligand system in the pathogenesis of MAIDS.
Thy‐1‐mediated activation of rat basophilic leukemia cells does not require co‐expression of the high‐affinity IgE receptor
Authors: Dráberová Lubica , Dráber Petr
Published: March 2007

The glycosylphosphatidylinositol (GPI)‐anchored protein Thy‐1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia cells, RBL‐2H3. Antibody‐mediated aggregation of Thy‐1 induces in these cells release of secretory components; so does aggregation of the receptor with high affinity for IgE (FcϵRI). To examine whether there is any relationship between Thy‐1‐ and FcϵRI‐mediated activation, we have isolated from mutagenized RBL‐2H3 cells a variant cell line deficient in the expression of surface FcϵRI, and analyzed its ability to be activated by an antibody to Thy‐1. Northern and immuno‐blot analyses revealed that the variant cells were deficient in the expression of a structural or a regulatory gene for FcϵRI γ subunit. The cells did not respond by release of secretagogues and protein‐tyrosine phosphorylation to IgE and antigen and anti‐FcϵRI monoclonal antibody (mAb) but their response to anti‐Thy‐1.1 mAb and calcium ionophore A23187 was retained. Transfection of the cloned FcϵRI γ subunit into the variant cells restored the surface expression of FcϵRI and responsiveness to both the antigen and anti‐FcϵRI mAb but had no effect on responsiveness to anti‐Thy‐1 mAb. The combined data indicate that aggregation of surface Thy‐1 glycoproteins activates a metabolic pathway which is independent of the presence of FcϵRI γ subunit and surface expression of FcϵRI.
Molecular characterization of human CD94: A type II membrane glycoprotein related to the C‐type lectin superfamily
Authors: Chang Chiwen , Rodríguez Antonio , Carretero Marta , López‐Botet Miguel , Phillips Joseph H. , Lanier Lewis L.
Published: March 2007

Natural killer (NK) cells preferentially express several genes of the C‐type lectin superfamily which have been implicated in the regulation of NK cell function. We demonstrate that CD94 is a type II membrane protein encoded by a unique gene of the C‐type lectin superfamily. While homology of CD94 with the NK cell‐associated NKR‐P1 and NKG2 C‐type lectin genes is limited to the structural motifs conserved in the carbohydrate recognition domain, all of these genes are on human chromosome 12, the syntenic of mouse chromosome 6, where genes of the NK complex (NKR‐P1 and Ly‐49) are located. An unexpected feature of CD94 is the essential absence of a cytoplasmic domain, implying that association with other receptors may be necessary for the function of this molecule.
CD4 and CD45 regulate qualitatively distinct patterns of calcium mobilization in individual CD4+ T cells
Authors: Leitenberg David , Constant Stephanie , Lu Dan Dan , Smith Brian R. , Bottomly Kim
Published: March 2007

An early consequence of T cell activation is an increase in intracellular calcium concentration. Recent advances in video laser microscopic techniques enable the examination of individual cells over time following stimulation. Such studies have revealed that cells can undergo qualitatively distinct patterns of calcium mobilization, suggesting that different patterns of calcium flux may be associated with different signaling pathways and may differentially affect late events in cell activation. In this report, we identify distinct patterns of calcium mobilization in CD4+ T cells following the antibody‐mediated cross‐linking of either CD3 or CD4, or following the cross‐linking of both CD3 and CD4 simultaneously. These effects can be further modified by the cross‐linking of CD45. We find that antibody cross‐linking of CD3 alone induces a single spike in the vast majority of cells shortly after the addition of the cross‐linking antibody. In contrast, cross‐linking CD4 alone induces a delayed pattern of repetitive calcium spikes which are decreased in amplitude compared to CD3 cross‐linking. Simultaneous cross‐linking of CD3 and CD4 induces a sustained increase in intracellular calcium mobilization which is dependent on the presence of extracellular calcium. This sustained increase in intracellular calcium concentration is also seen following physiologic cross‐linking of CD3 and CD4 after T cell interaction with specific antigen and antigen‐presenting cells. Finally, the simultaneous cross‐linking of CD45, CD3 and CD4 abrogates the sustained increase in calcium seen following CD3 and CD4 cross‐linking. These results suggest that the qualitative nature of T cell receptor signaling can be modulated by the molecular association of other signaling molecules, which may be part of the T cell receptor complex or not.
Macrophage‐T cell interaction in experimental visceral leishmaniasis: failure to express costimulatory molecules on Leishmania‐infected macrophages and its implication in the suppression of cell‐mediated immunity
Authors: Saha Bhaskar , Das Gobardhan , Vohra Harpreet , Ganguly Nirmal K. , Mishra Gyan C.
Published: March 2007

The most important immunopathological consequence of infection with Leishmania seen in murine and human hosts is the suppression of T cell‐mediated immune responses to both mitogens and leishmanial antigens. It has been suggested that this suppression is mediated by macrophages, either by defective antigen processing and presentation or by the elaboration of suppressive mediators like prostaglandins. Optimum activation of T helper cells requires not only T cell receptor occupancy by the antigen‐Ia complex, but also costimulatory signals provided by the antigen‐presenting cells. We investigated the status of several costimulatory molecules on infected macrophages from both genetically susceptible BALB/c and resistant C57BL/6 mice. Our results demonstrate that upon parasitization, the macrophages become unable to deliver costimulatory signals to T helper cells, and that this effects is mediated by prostaglandins, as the inhibition of its synthesis by indomethacin recovered the defect. Upon infection with L. donovani, B7‐1 expression was decreased, while ICAM‐1 was marginally increased in BALB/c macrophages and there was no significant change in the expression of B7‐1 and ICAM‐1 in Leishmania‐infected C57BL/6 macrophages. Expression of VCAM‐1 did not change during infection. This selective alteration in the expression of costimulatory molecules on L. donovani‐infected BALB/c macrophages was caused by the living parasite, as shown by the fact that killing of the parasites by stibogluconate led to no alteration in the levels of costimulatory molecules. We found that the change in B7‐1 expression on the surface of infected macrophages resulted in the inhibition of delayed‐type hypersensitivity‐mediating functions of T helper cells from BALB/c mice. The results described in this study not only throw light on the possible mechanism of leishmanial pathogenesis, but also open up the possibility of immunotherapy of leishmaniasis by selective manipulation of costimulatory molecules.
Various V‐J rearrangement efficiencies shape the mouse λ B cell repertoire
Authors: Boudinot Pierre , Rueff‐Juy Dominique , Drapier Anne‐Marie , Cazenave Pierre‐André , Sanchez Pierre
Published: March 2007

The diversity of the B cell repertoire of Cx knockout mice is limited by the expression of four λ light chain types. Among the spleen B cells, λ1 is expressed by the majority (58%) of cells, and λ3 by the minority (8%), while λ2(V2) and λ2(Vx) are expressed in intermediate quantities (18% and 16%, respectively). To assess the influence of mechanistic pressures on the λ subtype distribution, the proportions of the different λ rearrangements were determined in various B cell subpopulations divided on the basis of the λ subtype expressed, and the VλJλ junction sequences were studied at different steps of B cell differentiation (pre‐B, immature and mature B cells). The data show that (1) the ratio of productive/non‐productive VJ junctions is determined by the nature of the λ segments that are rearranged as can be observed in the pre‐B cells, (2) V1‐J1 non‐productive rearrangements are often found in the λ1‐negative B cells in the periphery, and (3) V1J3 junctions are often non‐productive regardless of the nature of the cells analyzed. Our results, therefore, suggest that a strong probability of initiating a V1‐J1 rearrangement and a weak probability of giving a productive V1J3 junction are responsible for the λ1 dominance and the λ3 under‐expression, respectively. The intermediate proportion of λ2(V2) subtype is most likely due to a probability of obtaining a productive joint that is better than that for V1J3 and a probability of initiating a rearrangement that is lower than that for V1J1. However, the λ2(Vx) cell proportion cannot be determined only by these parameters.
The majority of murine VH12‐expressing B cells are excluded from the peripheral repertoire in adults
Authors: Ye Jian , McCray Suzanne K. , Clarke Stephen H.
Published: March 2007

We have previously demonstrated that at birth most productive (P) VH12 rearrangements in B10.H‐2aH‐4bp/Wts (2a4b) mice encode a ten‐amino acid CDR3, and that a significant fraction of the expected repertoire is absent. We have now examined the adult VH12 CDR3 repertoire involving all four JH gene segments in both peritoneum and spleen. Of the 74 P VH12 rearrangements from these tissues 67 encode a CDR3 of ten amino acids and include a Gly in the fourth position (designated 10/G4). Most of these rearrangements appear to derive from phosphatidylcholine (PtC)‐specific B cells, which also have a 10/G4 VHCDR3, since few 10/G4 P rearrangements were present in spleen cells depleted of PtC‐specific B cells. Thus, the VH12 B cell repertoire in adult mice is largely restricted to the use of a single CDR3 motif and to a single antigen specificity. This bias results from two selection events: (1) selective exclusion of most VH12 B cells from the peripheral repertoire, and (2) clonal expansion in the periphery of VH12 B cells that have a 10/G4 VHCDR3 and bind PtC. Analysis of VH12‐JH1 rearrangements in viable motheaten (mevJmev) mice, which have an abnormal B cell repertoire due to a defective phosphatase (Hcph) and have barely detectable numbers of PtC‐specific B cells, indicates that selective exclusion of VH12 B cells from the peripheral repertoire occurs normally, but that clonal expansion of 10/G4 VH12 B cells is minimal. This is evidence that the selective exclusion of VH12 B cells from the peripheral repertoire and the clonal expansion of VH12 B cells with a 10/G4 CDR3 are due to independent signaling events.
Intercellular adhesion molecule‐3 is the predominant co‐stimulatory ligand for leukocyte function antigen‐1 on human blood dendritic cells
Authors: Starling Gary C. , McLellan Alexander D. , Egner William , Sorg Rüdiger V. , Fawcett Jonathan , Simmons David L. , Hart Derek N. J.
Published: March 2007

Dendritic cells (DC) are potent stimulators of primary T lymphocyte responses to foreign antigen. The initial DC‐T lymphocyte interaction involves the binding of the adhesion molecule leukocyte function antigen‐1 (LFA‐1; CD11a/CD18) on the T lymphocyte to an intercellular adhesion molecule (ICAM) on the DC. Although blood and tonsil DC express ICAM‐1 (CD54) and ICAM‐2 (CD102) on their surface, anti‐ICAM‐1 and anti‐ICAM‐2 monoclonal antibodies (mAb) have little inhibitory activity on the DC‐stimulated mixed leukocyte reaction (MLR). We therefore examined the expression of the more recently identified LFA‐1 ligand, ICAM‐3 (CD50), in comparison to ICAM‐1 and ICAM‐2 on blood DC and sought a functional role for ICAM‐3 in DC‐mediated T lymphocyte responses. Resting blood DC expressed significantly more ICAM‐3 than ICAM‐1 or ICAM‐2 as assessed by flow cytometry. Treatment of resting DC with interferon‐γ led to increased expression of ICAM‐1; however, ICAM‐2 and ICAM‐3 levels remained relatively constant. Solid‐phase recombinant chimeric molecules ICAM‐1‐, ICAM‐2‐ and ICAM‐3‐Fc were able to co‐stimulate CD4+T lymphocyte proliferation in conjunction with suboptimal solid‐phase CD3 mAb 64.1. However, the anti‐ICAM‐3 mAb CAL 3.10 inhibited a DC‐stimulated MLR to a greater extent than anti‐ICAM‐1 or anti‐ICAM‐2 reagents and appeared to act by blocking the DC ICAM‐3‐ T lymphocyte LFA‐1 interaction. As ICAM‐3 is the predominant LFA‐1 ligand on resting blood DC, we postulate that DC may utilize ICAM‐3 for initial DC‐T lymphocyte interactions, and that ICAM‐1, which is up‐regulated upon DC activation, and/or ICAM‐2, may contribute to DC migration or later phases of the T lymphocyte activation process.