bluuebunny/crossref_metadata_2025 huggingface dataset:
A CD44 monoclonal antibody differentially regulates CD11a/CD18 binding to intercellular adhesion molecules CD54, CD102 and CD50
Authors: Vermot‐Desroches Claudine , Wijdenes John , Valmu Leena , Roy Catherine , Pigott Rod , Nortamo Pekka , Gahmberg Carl G.
Published: March 2007

We have made a monoclonal anti‐CD44 antibody which is able to activate the leukocyte integrin CD11a/CD18. Activated T cells strongly aggregated, and the aggregation was shown to be intercellular adhesion molecule (ICAM)‐1 (CD54) and ICAM‐2 (CD102) dependent. Using purified ICAM coated on plastic, only binding to ICAM‐1 was increased by the CD44 antibody, whereas activation by phorbol ester increased binding to both ICAM‐1 and ICAM‐3. The binding to ICAM‐2 was not affected by either treatment. These findings show that the CD11a/CD18 integrin can be activated in a ligand‐specific manner by engagement of CD44.
Activation and hapten inhibition of mast cells sensitized with monoclonal IgE anti‐penicillin antibodies: evidence for two‐site recognition of the penicillin derived determinant
Authors: Fernandez Mercedes , Warbrick E. Victoria , Blanca Miguel , Coleman John W.
Published: March 2007

We utilized an in vitro mast cell activation assay and hapten inhibition of mediator release to characterize the fine specificity of two IgE anti‐penicillin monoclonal antibodies (mAb). Cultured mouse mast cells were passively sensitized with IgE mAb anti‐benzylpenicillin (BP) or anti‐amoxicillin (AX) and challenged with a range of penicillin‐human serum albumin (HSA) conjugates. Mast cells sensitized with IgE anti‐BP degranulated in response to BP‐HSA, but not to AX‐HSA or ampicillin(AMP)‐HSA, whereas mast cells sensitized with IgE anti‐AX responded to AX‐HSA but not to BP‐HSA or AMP‐HSA. Because BP, AX and AMP differ chemically only in the structure of their side chain, these results show that this part of the drug molecule is essential for recognition by IgE antibody. Unexpectedly, although IgE‐sensitized mast cells responded to only one penicillin in protein‐conjugated form, antigen‐induced degranulation was inhibited by the monomeric derivative of more than one penicillin. Furthermore, antigen activation of IgE‐sensitized cells was inhibited, although less potently, by haptens representative of the specific penicillin side chain or the binuclear portion of the drug molecule. These patterns of recognition and hapten inhibition were also seen in solid‐phase enzyme‐linked immunosorbent assay (ELISA), although all haptenic inhibitors were approximately 100 times less potent in the ELISA compared to the mast cell assay. To explain these findings we propose a model in which IgE binding to penicillin‐protein antigen is dependent on recognition of two distinct epitopes on the drug molecule: the first comprising the side chain, and the second comprising the binuclear portion plus the proximal region of the side chain. This two‐site hypothesis provides a generally applicable model of antibody recognition of penicillins and provides a rational basis for understanding the specificity and cross‐reactivity of IgE‐mediated allergic reactions to penicillins.
Involvement of NAK‐1, the human nur77 homologue, in surface IgM‐mediated apoptosis in Burkitt lymphoma cell line BL41
Authors: Mapara Markus Y. , Weinmann Pamela , Bommert Kurt , Daniel Peter T. , Bargou Ralf , Dörken Bernd
Published: March 2007

The induction of apoptosis via surface IgM (sIgM) in immature B cells requires de novo transcription. To investigate the regulation of activation‐induced cell death (AICD) in B cells we used a cell line model consisting of an Epstein‐Barr virus‐negative Burkitt lymphoma cell line (BL41), which is highly sensitive, and a subclone which is resistant to sIgM‐mediated apoptosis (BL41/B5). Resistance in this cell line was not due to down‐regulation of sIgM or functional impairment in signal transduction of the surface Ig complex. The zinc finger transcription factor nur77 has been implicated to play an important role in CD3‐mediated apoptosis in murine T cells. We were able to demonstrate that surface IgM ligation and subsequent apoptosis in BL41 cells is associated with a concomitant induction of NAK‐1, the human nur77 homologue. Induction of NAK‐1 mRNA and DNA binding activity in the nucleus could be readily observed by means of Northern blot and electrophoretic mobility shift assay, respectively. In contrast, the resistant clone BL41/B5 did not show any NAK‐1 expression upon stimulation. This suggests a role for NAK‐1 in sIgM‐mediated apoptosis of immature B cells.
Evidence for Th2 cell‐mediated suppression of antibody responses in transgenic, beef insulin‐tolerant mice
Authors: Teng Yen‐Tung , Gorczynski Reginald M. , Iwasaki Shinji , Williams Douglas B. , Hozumi Nobumichi
Published: March 2007

Clonal deletion, anergy and suppression have all been considered mechanisms of immunological tolerance. Although adoptive transfer of immunosuppression has been shown to occur in the periphery, particularly for transplantation tolerance, it has proven difficult to characterize this phenomenon further, due to the lack of suppressor T cell clones. To characterize tolerance towards a physiological soluble antigen, we constructed beef insulin (BI) transgenic (Tg) BALB/c (H‐2d) mice, in which the BI transgene is expressed in pancreatic β cells. These Tg mice were tolerant to BI immunization at the level of both humoral and cell‐mediated immune responses. Adoptive transfer of splenocytes from Tg mice into normal syngeneic BALB/c mice demonstrated that the reduction in antibody production is regulated by transferred T cells. The cytokine profile of T cell clones obtained after selection in vitro demonstrated dominant Th1 clones from normal non‐Tg mice and dominant Th2 clones from Tg mice. Some Th2 clones (CD4+) from Tg mice produced significant suppression of antibody production after adoptive transfer into normal syngeneic BALB/c mice. These data confirm the existence of Th2 regulatory T cells in vivo in a model of peripheral tolerance to a physiological soluble antigen as a potential mechanism for self tolerance.
Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells
Authors: Koh Dow‐Rhoon , Ho Alexandra , Rahemtulla Amin , Fung‐Leung Wai‐Ping , Griesser Henrik , Mak Tak‐Wah
Published: March 2007

MRL/lpr mice develop a systemic autoimmune disease similar to systemic lupus erythematosus in humans. The mice show progressive lymphadenopathy due to the accumulation of an unusual population of CD4−8−(DN) B220+ αβ+ T cells. We bred MRL/lpr mice with mice lacking CD4+ or CD8+ T cells by gene targeting via homologous recombination in embryonal stem cells to determine the roles of these cells in the autoimmune disease. No difference in survival or autoantibody levels was noted between CD8‐/‐ lpr and littermate controls. Interestingly, these CD8‐/‐ lpr mice have a reduced level of B220+ DN T cells despite the fact that the degree of lymphadenopathy was unaltered. CD4‐/‐ lpr mice had a diminished autoimmune disease with a reduction in autoantibody production and skin vasculitits, and increased survival compared to littermate controls. However, CD4‐/‐ lpr mice had an enhanced splenomegaly that developed massively by 16–20 weeks of age (5 to 8 greater than lpr control mice) due to the accumulation of DN B220+ T cells. In addition, there were no differences in peripheral lymph node enlargement, although the proportion of DN B220+ T cells was about twofold higher in the CD4‐/‐ lpr mice. These cells were phenotypically identical to the DN population in control lpr mice, indicating that the accumulating DN T cells can be dissociated from the autoimmune disease in these mice. Collectively, our results reveal that the autoimmune disease is dependent on CD4+, but not CD8+ T cells, and that many of the B220+ DN T cells traverse a CD8 developmental pathway.
Extensive allelic sequence variation in the J region of the human immunoglobulin heavy chain gene locus
Authors: Mattila Petri S. , Schugk Jan , Wu Hongyan , Mäkelä Olli
Published: March 2007

During the initial stages of B lymphocyte differentiation heavy chain variable (VH), diversity (DH) and joining (JH) gene segments recombine to form a functional heavy chain variable region (VDJ) gene. Evidence for genetic polymorphism of the human JH gene segments has been obtained from mature rearranged VDJ sequences. We conducted an analysis of the published rearranged JH gene sequences and found that the JH alleles present in the two published germ‐line JH region sequences were rare (approx. 2%) in the rearranged sequences. As an attempt to explain this discrepancy a 2.5‐kb strech of DNA containing all the six heavy chain JH region genes and the most 3' DH gene segment, DHQ52, was amplified by the polymerase chain reaction from 39 individuals and analyzed for restriction fragment length polymorphism. Five new JH region haplotypes were found and sequenced. These new haplotypes contained the coding segment alleles that were frequent in antibody genes. Surprisingly, a high number of interallelic differencies in the non‐coding sequence was found between the new and the two previously published haplotypes implying that the haplotypes had been separated early in evolution. In this respect the JH locus resembles HLA loci.
Evidence that CD4+, but not CD8+ T cells are responsible for murine interleukin‐2‐deficient colitis
Authors: Simpson Stephen J. , Mizoguchi Emiko , Allen Deborah , Bhan Atul K. , Terhorst Cox
Published: March 2007

Mice deficient in interleukin‐2 production (IL‐2null mice) develop colonic inflammation closely resembling ulcerative colitis in humans. Although this disease is marked by substantial infiltration of the colon by CD8+ and CD4+ T lymphocytes, no function has yet been assigned to these T cell subsets in the development of colitis in the IL‐2null mouse. For the present study, we investigated the involvement of T lymphocytes in the onset of colitis in IL‐2null mice, and examined the possible role played by cytotoxic T cells. Both lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) of the colon of IL‐2null mice were potently cytotoxic ex vivo in short‐term redirected cytotoxic lymphocyte (CTL) assays. In contrast, colonic T cells of wild‐type animals showed little or no constitutive cytotoxic T cell activity. Colonic CTL were detectable prior to the appearance of disease in IL‐2null animals and CTL activity was confined to the TcRαβ, rather than to the TcRγδ IEL subset. IL‐2null animals crossed with major histocompatibility complex class I‐deficient mice [IL‐2null × β2 microglobulin (β2mnull] mice also developed colitis, which appeared even earlier than in most IL‐2null mice. These findings suggest that neither CD8+ IEL nor LPL were causal in the onset of colitis in IL‐2null animals. In IL‐2null × β2mnull mice, an ulcerative colitis‐like disease was evident from histological studies and immunohistological staining which showed very large numbers of CD4+ lymphocytes within the intestinal mucosa. Significant ex vivo killing by CD4+ T cells was observed in IL‐2null × β2mnull animals, although this required an extended incubation time compared to colonic CD8+ T cells. Peripheral as well as colonic CD4+ T cells in IL‐2null and IL‐2null × β2mnull animals, were activated as judged by their cell surface phenotype (CD45RBlo, L‐selectinlo and CD69+). In light of these findings, we propose that infiltrating CD4+, but not CD8+ T cells are central to the inflammation observed in the intestinal mucosa in IL‐2null colitis.
Cytotoxic T lymphocytes raised against a subdominant epitope offered as a synthetic peptide eradicate human papillomavirus type 16‐induced tumors
Authors: Feltkamp Mariet C. W. , Vreugdenhil Gienke R. , Vierboom Michel P. M. , Ras Elisabeth , van der Burg Sjoerd H. , Schegget Jan Ter , Melief Cornelis J. M. , Kast W. Martin
Published: March 2007

Previously, we have shown that immunization with human papillomavirus (HPV) type 16‐derived cytotoxic T lymphocyte (CTL) epitope E7 49–57 (RAHYNIVTF) renders C57BL/6 mice insensitive to tumors formed by HPV16‐transformed cells. In this study, we provide evidence that E7 49–57 is expressed as a subdominant CTL epitope on HPV16‐transformed C57BL/6 cells. Using acid peptide elution, it is shown that HPV16‐transformed cells express another CTL epitope, besides E7 49‐57, which appears to be dominant. We demonstrate that a CTL line raised against the subdominant CTL epitope, offered as synthetic peptide E7 49–57, eradicates established HPV16‐induced tumors in mice. Our data show that synthetic peptide‐induced CTL can be applied successfully in vivo against (virus‐induced) tumor, and emphasize that subdominant CTL epitopes are useful targets for immunotherapy. Furthermore, it is illustrated for the first time that HPV16‐specific CTL interfere directly with HPV16‐induced tumors.
T cell development and repertoire of mice expressing a single T cell receptor α chain
Authors: Brändle Daniel , Brduscha‐Riem Karin , Hayday Adrian C. , Owen Michael J. , Hengartner Hans , Pircher Hanspeter
Published: March 2007

We examined T cell development and T cell repertoire in transgenic mice expressing a single T cell receptor (TCR) α chain derived from the H‐2Db ‐lymphocytic choriomeningitis virus (LCMV)‐specific cytolytic T lymphocyte (CTL) clone P14. To generate these α P14 mice, mice transgenic for the P14 TCR α chain were backcrossed to TCR α‐deficient mice. Thymi from α P14 mice exhibited a marked decrease of mature CD4+8− and CD8+4− single‐positive thymocytes comparable to thymi from TCR α‐deficient mice. Correspondingly, the number of peripheral T cells was reduced in the CD4 (tenfold) and in the CD8 (twofold) subsets when compared to normal mice. T cells from α P14 mice generated a primary anti‐LCMV CTL response when stimulated in vitro with LCMV in contrast to normal mice which require priming in vivo; elimination of LCMV in vivo was, however, not improved. Flow cytometric analysis of T cells with Vβ‐specific antibodies showed a diverse endogenous TCR Vβ repertoire. Functional analysis of the T cell repertoire, however, revealed a strongly reduced (30‐fold) allogeneic and the absence of a vesicular stomatitis virus‐specific CTL response and an impaired ability to provide T cell help for antibody isotype switching. Thus, T cell selection in the thymus was impaired and the T cell repertoire was limited in mice expressing only one type of TCR α chain.
Comparison of CD28‐B7.1 and B7.2 functional interaction in resting human T cells: Phosphatidylinositol 3‐kinase association to CD28 and cytokine production
Authors: Ghiotto‐Ragueneau Marguerite , Battifora Michela , Truneh Alemsedeg , Waterfield Michael D. , Olive Daniel
Published: March 2007

CD28 is a 44‐kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter‐receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these costimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3‐kinase (PI3‐K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28‐transfected cells or pre‐activated T cells. In addition, recent reports propose that CD28‐B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28‐B7.1 and CD28‐B7.2 interactions induce the association of PI3‐K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine‐phosphorylated CD28 and does not require pre‐activation by CD3‐T cell receptor. Worthmannin, a specific inhibitor of PI3‐K enzymatic activity within the nanomolar range also inhibits the interleukin‐2 production induced by costimulation mediated by either the B7.1‐ and B7.2‐transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC50 of wortmannin being 25 and 110 nM, respectively, which could suggest differences in their activation of the T cell PI3‐K.
Modulation of expression of the anti‐inflammatory cytokines interleukin‐13 and interleukin‐10 by interleukin‐3
Authors: Marietta Eric V. , Chen Yiyou , Weis John H.
Published: March 2007

Interleukin (IL)‐4, IL‐10 and IL‐13 are cytokines with potent anti‐inflammatory activities. Prevention of pathological inflammation at mucosal surfaces appears to be due, in part, to the presence of these cytokines. One potential source for these cytokines is the mast cell which resides at mucosal surfaces. Demonstrated in this report are the findings that bone marrow‐derived mucosal‐like mast cells constitutively expressed IL‐13 whereas bone marrow‐derived connective tissue‐like mast cells demonstrated IL‐13 transcription only after FcεRI‐mediated activation or the addition of exogenous IL‐3. A similar pattern of expression of IL‐10 by these mast cell types was also evident and matches that of IL‐4 previously reported. Intracellular cytokine staining indicated that IL‐10 protein is constitutively expressed by the bone marrow‐derived mucosal‐like mast cells but is only evident in the bone marrow‐derived connective tissue‐like mast cells after induction with IL‐3. The increase of IL‐13 and IL‐10 transcripts in the connective tissue‐like mast cells following IL‐3 treatment is not mast cell specific, in that splenic and bone marrow cells also demonstrated the same phenomenon. These data suggest that mucosal mast cells may have a constitutive repertoire of Th2 cytokines with potential anti‐inflammatory activity, while connective tissue mast cells may not. However, production of such cytokines can be induced in the connective tissue mast cell and other cell types of the immune response by the addition of IL‐3.
Anti‐CD5 extends the proliferative response of human CD5+ B cells activated with anti‐IgM and interleukin‐2
Authors: Jamin Christophe , Le Corre Rozenn , Lydyard Peter M. , Youinou Pierre
Published: March 2007

The CD5 T cell glycoprotein which is expressed by a subset of B cells has been shown to be involved in T cell activation and proliferation. No similar studies, to date, have addressed the role of CD5 on the B cell subset. CD5+ and CD5− B cells were sorted and stimulated with anti‐CD5 monoclonal antibody (mAb) in vitro. The activation and proliferative responses of these two populations, as measured by analysis of proliferation marker, did not differ following anti‐μ and interleukin (IL)‐2 stimulation. The addition of anti‐CD5 did not change the responsiveness of such activated CD5+ B cells but resulted in a decrease in CD25 expression. Pre‐activation of B cells with phorbol 12‐myristate 13‐acetate, which increased CD5 expression, failed to alter the proliferative response of CD5+ B cells to anti‐μ and IL‐2 with or without addition of anti‐CD5 mAb. Anti‐μ and IL‐2 treatment of CD5+ cells resulted in optimal proliferation measured at day 3 which decreased by day 6. However, addition of anti‐CD5 mAb at day 3 prevented this decline in proliferative response. This dose‐dependent effect was observed only when the anti‐CD5 mAb was presented to the B cells in cross‐linked form. Co‐stimulation of CD5 did not lower the threshold of antigen to which the B cells responded. Taken together, these data support a functional role for CD5 on B cells acting as an accessory signal, following their primary activation through the B cell receptor complex and highlight differences in the role of CD5 associated with the T cell receptor complex.
Proximity relationships between the type I receptor for Fcεe (FcεeRI) and the mast cell function‐associated antigen (MAFA) studied by donor photobleaching fluorescence resonance energy transfer microscopy
Authors: Jürgens Ludger , Arndt‐Jovin Donna , Pecht Israel , Jovin Thomas M.
Published: March 2007

Clustering of the mast cell function‐associated antigen (MAFA) on the surface of rat mucosal type mast cells line 2H3 (RBL‐2H3) leads to suppression of the secretory response induced by the type I Fcεe receptor (FcεeRI). In order to establish a possible association between MAFA and FcεeRI we measured fluorescence resonance energy transfer (FRET) between the MAFA‐specific monoclonal antibody (mAb) G63 and FcεeRI‐bound ligands as well as between FcεeRI‐bound ligands themselves using the donor photobleaching FRET (pbFRET) technique. Average FRET efficiencies between 6 and 9% were determined after low‐temperature incubation with fluorescent dye conjugated mAb G63 bound to MAFA (donor) and IgE bound to FcεeRI (acceptor) on RBL‐2H3 cells. Subsequent cross‐linking of IgE by a polyvalent antigen caused no change in FRET efficiencies. These results suggest that the MAFA is located in the vicinity of the FcεeRI on resting cells, and that clustering of the FcεeRI leads to no significant change in the proximity of the two molecular species. In view of the sequence motif identified in the cytosolic tail of the MAFA and the observed changes in its phosphorylation upon antigen stimulation (Guthmann et al., Proc. Natl. Acad. Sci. USA 1995, 92: 9397–9401), the present study suggests that the secretory response inhibition by MAFA interferes with the signal transduction cascade initiated via the FcεeRI. An additional finding was that clustering of the FcεeRI by antigen showed a clear increase in the efficiency of FRET between FcεeRI‐bound IgE molecules conjugated with fluorescent donor and acceptor.
Expression and function of Fas antigen on activated murine B cells
Authors: Wang Jiyang , Taniuchi Ichiro , Maekawa Yoichi , Howard Maureen , Cooper Max D. , Watanabe Takeshi
Published: March 2007

We have studied the expression and function of Fas antigen on murine B lymphocytes. While Fas was present on only a few B cells in the bone marrow, spleen, lymph node or peripheral blood, its expression could be strongly up‐regulated by stimulation with soluble CD40 ligand (CD40L). Treatment with anti‐IgM and interleukin‐4 (IL‐4) alone did not induce significant Fas expression but enhanced CD40L‐mediated up‐regulation of Fas expression. The T cell‐derived signal via CD40 is therefore a potent inducer of Fas expression by B lymphocytes. The sensitivity to Fas‐mediated apoptosis was found to depend on the duration of B cell activation. B cells activated for 1 day were resistant to Fas‐mediated cell death, whereas B cells activated for 3 days were relatively sensitive. Interestingly, different sensitivity to Fas‐mediated death signal was observed in 2‐day activated B cells. It was found that B cells stimulated with CD40 L alone were more sensitive to Fas‐mediated apoptosis than were cells stimulated with CD40L plus anti‐IgM or IL‐4, and in particular, the combination of the two. The greater sensitivity exhibited by B cells stimulated with CD40L alone seems to be related to limited activation of these cells in the absence of additional stimulation. Co‐stimulation of B cells in the presence of CD40L and anti‐Fas antibody resulted initially in activation of B lymphocytes, as reflected by the expression of activation markers and cell growth, but this was followed by growth inhibition and cell death. The data demonstrate that the B cell response can be regulated positively and negatively by signaling through CD40 and Fas antigens, respectively.
Inhibition of murine B1 lymphocytes by interleukin‐12
Authors: Vogel Laura A. , Lester Terry L. , Van Cleave Victor H. , Metzger Dennis W.
Published: March 2007

B1 cells are a subset of B lymphocytes found in many spectes and are implicated in the development of autoimmunity. B1 cells have previously been shown to be suppressed by the T helper (Th)1 cytokine interferon (IFN)‐γ, and to be stimulated by the Th2 cytokines interleukin (IL)‐2, IL‐4, IL‐5 and IL‐10. To examine further the interactions of B1 cells and Th1 cells, we have now tested the effects of the Th1 cell‐inducing cytokine IL‐12 on murine B1 cells. BALB/c mice were immunized with phosphorylcholine conjugated to keyhole limpet hemocyanin (PC‐KLH) and simultaneously treated with 1 μg recombinant murine IL‐12 for 3 consecutive days. In addition to altering the isotype and idiotype distribution of anti‐PC antibodies, IL‐12 treatment was found to cause a loss of peritoneal, but not splenic B lymphocytes in immunized mice. B cell depletion required exposure to IL‐12 plus antigenic stimulation. Levels of peritoneal B lymphocytes were fully restored by day 45, but the majority of these cells belonged to the B2 subset. Additionally, proliferation of B1 cells in vitro induced by IL‐5 was substantially inhibited by IL‐12. IL‐12 itself had no effect on viable cell recovery of peritoneal cells (PeC) cultured in vitro, but viable cell recovery was significantly decreased in PeC cultured with IL‐5 plus IL‐12. These results show that IL‐12 causes the loss of murine peritoneal B1 cells and suggest that treatment with this cytokine may be useful for disease conditions that involve B1 cell dysfunction.
Interleukin‐2 receptor common γ‐chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: Selective induction of anti‐apoptotic (bcl‐2, bcl‐xL) but not pro‐apoptotic (bax, bcl‐xS) gene expression
Authors: Akbar Arne N. , Borthwick Nicola J. , Wickremasinghe R. Gitendra , Panayiotidis Panayiotis , Pilling Darrell , Bofill Margarita , Krajewski Stanislaw , Reed John C. , Salmon Mike
Published: March 2007

Cytokine deprivation from activated T cells leads to apoptosis associated with down‐regulation of the bcl‐2 gene product. It is not clear, however, how cytokines other than interleukin‐2 (IL‐2) may affect this process and regulate the involvement of other apoptosis‐modulating genes. We show that a group of cytokines including IL‐2, IL‐4, IL‐7 and IL‐15, which can all signal through the γ chain of the IL‐2R (IL‐2Rγ), prevent the apoptosis of IL‐2‐deprived activated T cells. This rescue involves the induction of the anti‐apoptosis genes (bcl‐2 and bcl‐xL), but causes little change in expression of bax and bcl‐xS, which promote apoptosis. Furthermore, the prevention of apoptosis and induction of proliferation by the common γ chain cytokines can be dissociated. Thus, when proliferation is blocked, the common γ chain cytokines still induce up‐regulation of bcl‐2 relative to bax and retard apoptosis. These cytokines can thus regulate the persistence or removal of effector T cells by coordinating the balance between genes which promote and those which inhibit apoptosis, events which are probably mediated at least in part by signals through the common γ chain. These data also implicate inappropriate T cell apoptosis resulting from a dysfunctional common γ‐chain as part of the pathophysiological defect in patients with X‐linked severe‐combined immunodeficiency (SCID).
B7‐1 synergizes with interleukin‐12 in interleukin‐2 receptor α expression by mouse T helper 1 clones
Authors: Igarashi Osamu , Yanagida Tetsuya , Azuma Miyuki , Okumura Ko , Nariuchi Hideo
Published: March 2007

Expression of interleukin‐2 receptor α (IL‐2Rα) is critical to induce interleukin (IL)‐2‐dependent proliferation of T helper (Th)1 clones. The IL‐2Rα expression of Th1 clones is known to be up‐regulated by IL‐12. Co‐stimulation via CD28/CTLA‐4 is also known to be important for efficient activation of CD4+ T cells. In the present experiments, IL‐12‐induced enhancement of IL‐2Rα expression of Th1 clones stimulated with B cells as antigen‐presenting cells (APC) is suppressed by the addition of anti‐B7‐1. To analyze the mechanism, Th1 clones were stimulated with immobilized anti‐CD3 plus IL‐12 in the presence or absence of chinese hamster ovary cells that express mouse B7‐1 (B7‐1CHO) and the enhancement of IL‐2Rα expression induced by the co‐stimulation was analyzed. The results of these experiments indicate that B7‐1 synergizes with IL‐12 in IL‐2Rα expression of the Th1 clone stimulated with anti‐CD3, although B7‐1CHO alone did not enhance IL‐2Rα expression of the clones. B7‐1 stimulation is not mediated by the enhancement of IL‐2 production: B7‐1 enhancement of IL‐2Rα expression was FK506 resistant, while the inclusion of FK506 abrogated IL‐2 production of the Th1 cells. B7‐1 co‐stimulation did not stabilize IL‐2Rα mRNA, but did synergize with IL‐12 to enhance IL‐2Rα mRNA transcription.
Neonatally tolerant rats actively eliminate donor‐specific lymphocytes despite persistent chimerism
Authors: Cutler Antony J. , Bell Eric B.
Published: March 2007

Rats from the allotype‐marked PVG‐RT7b and PVG‐RT1u‐RT7b strains were injected at birth with semi‐allogeneic F1 bone marrow (BM) cells from athymic nude rats (PVG‐rnu/rnu x PVG‐RT1u‐rnu/rnu) to induce neonatal tolerance. As adults, 97% of the animals accepted donor‐specific allogeneic skin grafts and a majority (65%) of rats were chimeric, expressing the major histocompatibility complex class I and allotype marker of the donor strain. Similar results were obtained when PVG‐RT1u‐RT7b rats were injected at birth with fully allogeneic PVG‐rnu/rnu nude BM cells: as adults, 94% accepted donor‐specific skin allografts and 76% of recipients were chimeric. Donor‐derived CD4T cells, CD8T cells and B cells were found in low numbers (< 2%) in peripheral blood of rats made tolerant by F1 BM cells. A large proportion of T cells bore the phenotype of recent thymic emigrants, suggesting that they were newly produced. All the evidence was consistent with clonal deletion tolerance, induced centrally within the thymus. The thymus was chimeric and thymocytes failed to respond in vitro to alloantigens of the donor‐specific haplotype; donor‐specific skin allografts survived indefinitely on athymic nude recipients reconstituted with CD4+CD8− thymocytes or peripheral CD4T cells from tolerant animals. The chimeric state was interesting, since the PVG and PVG‐RT1u rat strains contain a natural killer (NK) cell system that rapidly eliminates (within 24 h) intravenously injected allogeneic or semi‐allogeneic lymphocytes – a phenomenon known as allogeneic lymphocyte cytotoxicity or ALC. When neonatal tolerant rats were tested, the ALC index (a measure of cell killing) was unchanged in nonchimeric tolerant rats and significantly altered (reduced killing), but not abolished in chimeric animals. Hence, the injection of allogeneic BM cells which induced specific tolerance in the T cell population failed to tolerize the NK cell system, allowing the constant killing of newly produced donor‐derived lymphocytes and putting at risk the very survival of the allogeneic BM cells. This has interesting implications for clinical transplantation.
An HLA‐B35‐restricted epitope modified at an anchor residue results in an antagonist peptide
Authors: Dong Tao , Boyd Diana , Rosenberg William , Alp Nick , Takiguchi Masafumi , McMichael Andrew , Rowland‐Jones Sarah
Published: March 2007

Peptides associated with HLA‐B35 commonly have a proline or occasionally a serine residue in the P2 anchor position of the peptide, with a tyrosine at the C terminus. Based on this motif, we identified an octamer epitope from influenza A matrix protein which is presented by HLA‐B35. The requirements for MHC binding and T cell receptor contact have been analyzed using analogs of this peptide with substitutions at positions 1, 2, 4, 7 and 8. The natural epitope contains a serine residue at P2 of the peptide. Substitution of this residue with proline (the favored amino acid in this position in B35‐associated peptides) considerably enhances binding to HLA‐B35 in the T2‐B35 cell line, but the peptide is not recognized by the majority of CTL clones and can antagonize recognition of the index peptide. This suggests that a conservative substitution at the P2 anchor position results in a conformational change in the peptide‐MHC surface exposed to the T cell receptor.
IgD‐receptor up‐regulation on human peripheral blood T cells in response to IgD in vitro or antigen in vivo correlates with the antibody response to influenza vaccination
Authors: Swenson Christina D. , Cherniack E. Paul , Russo Carlo , Thorbecke G. Jeanette
Published: March 2007

Aged individuals (>65 years) were classified as antibody (Ab) responders on the basis that they showed increases to ⩾1:40 in serum Ab titers to all influenza virus strains present in the trivalent influenza vaccine within 4 weeks after immunization. The peripheral blood mononuclear cells (PBMC) from pre‐immunization samples of blood taken from seven Ab‐responders and seven Ab‐nonresponders were examined for their ability to exhibit up‐regulation of IgD‐receptor (IgD‐R) after exposure for 2 h to immobilized cross‐linked IgD, as shown by rosetting with IgD‐coated ox erythrocytes. The responsiveness to IgD was found to be predictive of the ability to produce Ab responses to viral protein Ag: the IgD‐R up‐regulation was > 5% in all Ab‐responders and < 4% in all the Ab‐nonresponders. In addition, there was an excellent correlation between mean Ab titers (to the three viruses in sera collected 4 weeks after immunization) and the percentage of IgD‐R+ cells obtained in response to IgD in PBMC from the same individual prior to immunization: p = 0.894.

Injection of influenza vaccine itself also induced IgD‐R on PBMC in vivo. The percentage of IgD‐R+ cells peaked after 24 h, was still detectable above background by day 7 or 14, and returned to pre‐injection levels by day 28 in young subjects and aged Ab‐responders, but not in Ab‐nonresponders. Similarly, purified peripheral blood T cells obtained from aged Ab‐responders exhibited IgD‐R upon immunization in vivo. These findings suggest that Ag injection causes rapid up‐regulation of IgD‐R by cross‐linking IgD in humans as well as in mice as shown previously. In analogy with results in mice, the present data are consistent with a role for IgD‐R+ T cells in the humoral response in man. Proliferative responses to influenza proteins in peripheral blood T cells from vaccinated individuals were found to peak on day 7 and were higher in Ab‐responders than in Ab‐nonresponders.
Interleukin‐6 induces tyrosine phosphorylation of the Ras activating protein Shc, and its complex formation with Grb2 in the human multiple myeloma cell line LP‐1
Authors: Neumann Carola , Zehentmaier Gundula , Danhauser‐Riedl Susanne , Emmerich Bertold , Hallek Michael
Published: March 2007

Like many other cytokines and growth factors, interleukin‐6 (IL‐6) activates p21ras. However, the precise biochemical mechanisms inducing this activation are unknown. Therefore, we investigated the effects of IL‐6 on some recently identified signaling intermediates, Shc (Src homology and collagen) and Grb2 (growth factor receptor bound protein 2), known to activate p21ras. In the multiple myeloma cell line LP‐1, IL‐6 stimulated the tyrosine phosphorylation of Shc in a time‐ and concentration‐dependent manner. This led to the complex formation of Shc with Grb2, an adaptor protein known to relocate a p21ras‐GDP exchange factor, Sos1 (Son‐of‐sevenless), to the cell membrane. Taken together, these findings suggest that IL‐6 might activate the Ras signaling pathway via tyrosine phosphorylation of Shc and subsequent recruitment of Grb2. Further studies will elucidate which of the IL‐6 receptor associated non‐receptor tyrosine kinases of the Src kinase or Janus kinase family, mediate these effects.
Expression of a mannose/fucose membrane lectin on human dendritic cells
Authors: Avraméas Alexandre , McIlroy Dorian , Hosmalin Anne , Autran Brigitte , Debré Patrice , Monsigny Michel , Roche Annie Claude , Midoux Patrick
Published: March 2007

Dendritic cells (DC) are the most efficient antigen presenting cells for T lymphocytes. CD1a+ CD14− DC with high antigen‐presenting capacities can now be obtained easily from adherent peripheral blood monocytes by culture in the presence of granulocyte/macrophage colony‐stimulating factor and interleukin‐4 (Sallusto et al., J. Exp. Med. 1994. 179: 1109). Human macrophages express a membrane lectin, or sugar‐specific receptor, which specifically mediates the binding and endocytosis of mannose‐ and fucose‐terminated glycoproteins and is involved in the phagocytosis of pathogens. A similar lectin activity was sought on cultured human DC using flow cytometry and confocal microscopy to detect binding and internalization of fluoresceinated neoglycoproteins [bovine serum albumin (BSA) substituted with sugar residues]. Several neoglycoproteins, especially α‐L‐fucosyl‐, α‐D‐mannosyl‐, N,N′‐di‐acetyl‐β‐chitobiosyl‐ and β‐D‐glucosyl‐BSA, were endocytosed by cultured human CD1a+ DC as well as by CD1a− CD14− cells which were also obtained in the culture. Fuc‐BSA and Man‐BSA had the same number of binding sites (1.7 × 106/cell) on CD1a+ DC, and bound with an affinity constant close to 107 l/mol. Inhibition experiments indicated that these two neoglycoproteins bound to the same membrane lectin. CD1a+ and CD1a− cells were both labeled by an antiserum specific for the human macrophage mannose receptor. The membrane lectin specific for mannose and fucose that is evidenced in these experiments on cultured DC may be similar to the macrophage membrane lectin or may share functional and structural properties with it.
Inhibition of I‐Ad‐, but not Db‐restricted peptide‐induced thymic apoptosis by glucocorticoid receptor antagonist RU486 in T cell receptor transgenic mice
Authors: Xue Yintong , Murdjeva Marianna , Okret Sam , McConkey David , Kiuossis Dimitris , Jondal Mikael
Published: March 2007

Thymocytes differentiate by positive and negative selection of immature CD4+CD8+T cells. Negative selection occurs by default or by high‐affinity recognition of peptides bound to proteins encoded by the major histocompatibility complex (MHC). MHC class I molecules are expressed on many different cell types, although at different levels, whereas MHC class II molecules are selectively expressed on thymic epithelial cells (TEC) and dendritic cells (DC). We investigated the role of the glucocorticoid receptor (GR) in thymic negative selection using the receptor antagonist RU486. Glucocorticoids (GC) are known to be potent inducers of apoptosis in CD4+CD8+thymocytes, and we have earlier shown that anti‐CD3‐induced thymic apoptosis can be blocked by RU486in vivo.We now show that anti‐CD3 induces thymic apoptosis in mice that have been adrenalectomized (ADX), and that RU486 inhibits anti‐CD3 antibody‐mediated thymocyte killing in newborn thymic organ cultures. Thymocyte apoptosis induced by ovalbumin peptide OVA323–339 treatment of mice transgenic for the DO11.10T cell receptor (TCR), which recognizes this peptide in the context of I‐Ad, was found to be inhibited by RU486. These mice responded to peptide treatment by an extensive activation of the peripheral immune system, which became lethal in 60% of the mice when accompanied by simultaneous RU486 treatment. In contrast, RU486 had no effect on thymic apoptosis induced by the influenza A nucleoprotein NP366–374 peptide, recognized in context of Db, in F5 TCR transgenic mice. We interpret the results to demonstrate that different deletion systems operate in the thymus. We propose that endogenous GC may be important for negative selection by default and by high‐affinity recognition of endogenous MHC‐presented peptides on TEC.
Binding of nucleosomes to a cell surface receptor: Redistribution and endocytosis in the presence of lupus antibodies
Authors: Kontouzov Sophie , Cabrespines Alban , Amoura Zahir , Chabre Henri , Lotton Chantal , Bach Jean‐François
Published: March 2007

In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV‐1, and questioned whether anti‐double‐stranded (ds)DNA and/or anti‐histone autoantibodies could recognized and influence the fate of cell surface‐bound nucleosomes. 125I‐labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration‐dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high‐affinity site with a Kd of ∼ 7nM and a low‐affinity site (Kd ∼ 400 nM) with a high capacity of 9 × 107 sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome‐derived dsDNA (180–200 bp) was found to compete for binding of 125I‐mononucleosomes on the low‐affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from 125‐I‐labeled cell lysates. Nucleosomes were found to precipitate not only the 180‐kDa dsDNA‐reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti‐dsDNA or anti‐histone antibodies (i.e. anti‐nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto‐antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome‐anti‐nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5–10 min which moved toward the perinuclear region by 20–30 min. By means of double‐fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome‐anti‐nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co‐distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome‐anti‐nucleosome complexes by disruption of the micro‐filament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti‐dsDNA and anti‐histone antibodies can form nucleosome‐anti‐nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.
Identification and characterization of a human CD4 silencer
Authors: Donda Alena , Schulz Manfred , Bürki Kurt , De Libero Gennaro , Uematsu Yasushi
Published: March 2007

Using transgenic mice, we have identified a human CD4 silencer contained within a 484‐bp fragment in the first intron of the CD4 gene. Further experiments have mapped a lineage‐specific silencing activity to a region of 190 bp. This region contains two protein‐binding sites detected by deoxyribonuclease I footprinting analyses. Tested in transient transfection assays, these two DNA elements showed significant silencing activity restricted to the CD8 phenotype. In CD4 cells, either no clear effect (FP I) or strong enhancing activity (FP II) was observed by transient transfection assays. Despite the lineage‐specific activity of these two elements, electrophoretic mobility shift assays (EMSA) showed similar levels of protein binding to the silencer element FP I in CD4 and CD8 T cells. Base substitutions in the FP I fragment abolished the silencing activity in transfected CD8 cells as well as the protein binding in EMSA, suggesting an important role of this protein‐DNA interaction in CD4 gene regulation.
Nitric oxide synthase: expression of the endothelial, Ca2+/calmodulin‐dependent isoform in human B and T lymphocytes
Authors: Reiling Norbert , Kröncke Rolf , Ulmer Artur J. , Gerdes Johannes , Flad Hans‐Dieter , Hauschildt Sunna
Published: March 2007

Nitric oxide (NO) is a pleiotropic mediator of a variety of cellular processes such as vasorelaxation, neurotransmission, and cytotoxicity. We studied the expression of the human endothelial, Ca2+/calmodulin‐dependent NO synthase (NOS) isoform (ecNOS) in highly purified human lymphocytes from peripheral blood and tonsillar tissue. ecNOS mRNA was detected in IgD+ or IgD− B cells, peripheral blood and tonsillar T cells. Upon stimulation, ecNOS mRNA expression decreased in all these lymphocyte subpopulations. Germinal center T cells and follicular dendritic cells did not express ecNOS mRNA either in an unstimulated or in a stimulated state. ecNOS expression by human lymphocytes was further substantiated by its mRNA detection in lymphoid cell lines such as Raji, Daudi, and Jurkat. By the use of a specific monoclonal antibody, ecNOS was shown to be present in T cells from peripheral blood and in various germinal center cells of frozen tonsillar sections. These data are the first to demonstrate the expression of the endothelial ecNOS at mRNA and protein level in human lymphocytes.
A functional analysis of recombinant soluble CD46 in vivo and a comparison with recombinant soluble forms of CD55 and CD35 in vitro
Authors: Christiansen Dale , Milland Julie , Thorley Bruce R. , McKenzie Ian F. C. , Loveland Bruce E.
Published: March 2007

The human cell surface complement regulatory proteins CD46 (MCP), CD55 (DAF) and CD35 (CR1) protect autologous cells from complement‐mediated damage by inhibiting C3 and C5 convertases. This regulatory potential has previously been exploited in the treatment of some models of inflammatory injury by the generation of recombinant soluble (rs) proteins, such as rsCD55 and rsCD35. More recently, we have shown that rsCD46 inhibits complement activation in the fluid phase. In this report, the ability of rsCD46, rsCD55 and rsCD35 to regulate human complement activation mediated by the classical and alternative pathways was directly compared. Regulation of the classical pathway in vitro was clearly demonstrated by all three soluble proteins; however, rsCD35 was a more effective inhibitor than either rsCD46 or rsCD55. A combination of rsCD46 + rsCD55 was more potent than either of these proteins alone. Cell lysis via alternative pathway activation in vitro was efficiently regulated by rsCD46 and rsCD35 to a similar extent, whereas rsCD55 was not as effective. Assays of rsCD46 in vivo have previously not been possible due to difficulties in expressing sufficient quantities of protein. This limitation has been overcome and now we report the ability of rsCD46 to inhibit immune complex‐mediated inflammation in a rat using the reverse passive Arthus reaction model. Administration of rsCD46 significantly reduced the size of lesion, and histological examination showed a reduction in inflammatory infiltrate and edema. These data suggest that rsCD46, in addition to rsCD55 and rsCD35, may be a useful therapeutic agent.
Generation of antigen‐presenting cells for soluble protein antigens ex vivo from peripheral blood CD34+ hematopoietic progenitor cells in cancer patients
Authors: Fisch Paul , Köhler Gabriele , Garbe Annette , Herbst Birgit , Wider Dagmar , Kohler Hubertus , Schaefer Hans E. , Mertelsmann Roland , Brugger Wolfram , Kanz Lothar
Published: March 2007

Peripheral blood CD34+ hematopoietic progenitor cells (PBPC) mediate hematopoietic reconstitution in cancer patients after autologous transplantation and can be expanded ex vivo in the presence of colony‐stimulating factors. This study shows that functionally active antigen‐presenting cells (APC) for soluble proteins are generated and expanded in these PBPC cultures. CD34+ cells were cultured ex vivo in medium containing stem cell factor, interleukin‐1β (IL‐1β), IL‐3, IL‐6, and erythropoietin (EPO). The cells from these cultures developed into very potent APC of tetanus toxid and purified derivative of tuberculin for autologous T cells in vitro. The antigen‐presenting capacity of these cells was maintained for at least 38 days of culture. These APC resembled immature cells of the myelomonocytic cell lineage by surface marker, immunocytochemistry and ultrastructural analysis. Such APC might be able to present antigens from certain tumors to the immune system.