bluuebunny/crossref_metadata_2025 huggingface dataset:
Bcl‐2 delays macrophage engulfment of human B cells induced to undergo apoptosis
Authors: Flora Pauline K. , Devitt Andrew , Johnson Gerald D. , Milner Anne E. , Gregory Christopher D.
Published: March 2007

The capacity to be recognized and engulfed by phagocytes is an important characteristic of cells dying by apoptosis. Phagocytosis of apoptotic cells occurs rapidly in vivo, probably prior to plasma membrane breakdown. While the molecular mechanisms mediating phagocytosis of apoptotic cells are beginning to be defined, little is yet known of the relationship between the cell‐death program itself and the surface changes on the dying cells that signal for engulfment. Here, we investigate to what extent the apoptosis repressor Bcl‐2 can modulate the recognition and phagocytosis of human B cells exposed to triggers of apoptosis. Burkitt lymphoma (BL)‐derived, Bcl‐2− B cells were induced into apoptosis either by the Ca2+‐ionophore ionomycin or by the inhibitor of protein synthesis cycloheximide. Apoptotic BL cells, but not viable BL cells, were recognized and phagocytosed by monocyte‐derived macrophages. bcl‐2‐transfected BL populations showed a reduced capacity both to undergo apoptosis in response to these inducing agents and to interact with macrophages. Like their Bcl‐2− counterparts, Bcl‐2+ BL cells interacted with macrophages only after activation of their apoptotic program as assessed by changes in nuclear morphology. These results demonstrate not only that continued protein synthesis in B cells undergoing apoptosis is not essential for their recognition by macrophages, but also that macrophage recognition of apoptotic B cells cannot be uncoupled from the cell‐death program that is controlled by Bcl‐2. In this respect, the behavior of B cells contrasts markedly with that of neutrophils in which Bcl‐2 has been reported to inhibit apoptosis without affecting phagocytic clearance (Lagasse and Weissman, J. Exp. Med. 1994. 179: 1047).
Major histocompatibility complex class I‐dependent skewing of the natural killer cell Ly49 receptor reportoire
Authors: Held Werner , Dorfman Jeffrey R. , Wu Ming‐Fan , Raulet David H.
Published: March 2007

Subsets of mouse natural killer (NK) cells express receptors encoded by the Ly49 gene family that recognize allelic determinants on major histocompatibility complex (MHC) class I molecules. Recognition of self class I molecules typically inhibits NK cell lytic function. The presence of NK cell subsets expressing receptors which are able to discriminate class I alleles raises the possibility that there exist mechanisms to coordinate the NK cell receptor repertoire with the class I molecules of the host. In the present study, we determined the effects of class I gene expression on the frequencies of NK cells expressing three different Ly49 receptors defined by monoclonal antibodies. We show here an MHC‐dependent skewing of NK cell subsets expressing multiple Ly49 receptors with specificity for self MHC. The results provide the first evidence that the frequencies of NK cells expressing different Ly49 receptors are determined by the host's MHC molecules. The results also extend previous findings that MHC class I expression influences the cell surface levels of each Ly49 receptor, suggesting an additional mechanism by which MHC molecules may influence the effective specificity of NK cells. Models to account for self tolerance and MHC‐controlled repertoire differences are discussed.
T cell receptor and CD8‐dependent tyrosine phosphorylation events in cytotoxic T lymphocytes: activation of p56lck by CD8 binding to class I protein
Authors: Anel Alberto , O'Rourke Anne M. , Kleinfeld Alan M. , Mescher Matthew F.
Published: March 2007

Tyrosine phosphorylation of proteins plays a central role in T cell activation. Mitogens or anti‐receptor antibodies have been employed to study these signaling events, but the extent to which these mimic receptor interactions with native ligands is unclear. Cytotoxic T lymphocytes can be activated for functional responses using purified, native class I ligands presented on a surface. Previous work showed that stimulation with fluid‐phase anti‐T cell receptor (TCR) monoclonal antibody (mAb) activates CD8 to mediate adhesion to class I proteins and that activated CD8 generates a co‐stimulatory signal upon binding to class I. Changes in tyrosine phosphorylation of substrates and activity of the p56lck kinase have now been examined in this two‐step process. The observed changes are small in comparison to those found using more potent nonphysiological stimuli, but may more accurately reflect the events required for activation of functional responses. Fluid‐phase anti‐TCR mAb caused increased tyrosine phosphorylation of a discrete subset of cellular substrates. Increased phosphorylation of additional substrates occurred upon CD8 binding to class I, resulting in a phosphorylation pattern comparable to that found in cells stimulated with class I alloantigen. Anti‐TCR mAb alone caused increased tyrosine phosphorylation of p56lck. When CD8 bound to class I, phosphorylation of p56lck decreased to below the basal level found in unstimulated cells, accompanied by a substantial increase in kinase activity. These results are consistent with the two‐step model for TCR activation of CD8/class I interactions and directly demonstrate that CD8 binding to class I leads to up‐regulation of p56lck activity.
CD40 is functionally expressed on human keratinocytes
Authors: Denfeld Ralf W. , Hollenbaugh Diane , Fehrenbach Alexandra , Weiss Johannes M. , von Leoprechting Achim , Mai Brigit , Voith Ursula , Schöpf Erwin , Aruffo Alejandro , Simon Jan C.
Published: March 2007

The CD40/gp39 pathway is known to be an important feature of B/T cell collaboration leading to T cell‐dependent activation, proliferation or differentiation of B cells. Additionally, CD40 is involved in the regulation of B cell survival and apoptosis. Recently, CD40 has been shown to be expressed functionally on non‐hematopoietic cells, i.e. endothelial cells. Here, we demonstrate that human keratinocytes (KC) cultured in vitro express CD40 constitutively. The surface expression of CD40 is markedly up‐regulated following stimulation with interferon (IFN)‐γ, but not with tumor necrosis factor‐α or interleukin (IL)‐1β. This process is regulated at the CD40 mRNA level as demonstrated by Northern blot analysis. Furthermore, ligation of CD40 via soluble gp39, the CD40 ligand, enhances intercellular adhesion molecule (ICAM)‐1 and Bcl‐x up‐regulation on IFN‐γ‐stimulated KC, but not lymphocyte function‐associated antigen (LFA)‐3, B7‐2, HLA‐DR, or Fas expression. The release of IL‐8 is also induced following CD40 ligation on KC. In psoriasis, a T cell‐mediated inflammatory skin disease, KC have a markedly enhanced expression of CD40. This expression co‐localizes with the expression of ICAM‐1, Bcl‐x, and an influx of CD3+ T cells. These findings suggest a functional role of CD40 on KC in inflammatory skin disorders such as psoriasis and could make a therapeutic intervention by disrupting the CD40/gp39 pathway an approach to consider in these inflammatory skin diseases.
Attenuation of collagen‐induced arthritis in mice by treatment with vector cells engineered to secrete interleukin‐13
Authors: Bessis Natacha , Boissier Marie‐Christophe , Ferrara Pascual , Blankenstein Thomas , Fradelizi Didier , Fournier Catherine
Published: March 2007

The anti‐inflammatory effects of the recently identified cytokine interleukin (IL)‐13 on collagen‐induced arthritis (CIA) was explored and compared to those of IL‐4 using systemic administration of these cytokines via two injections of xenogeneic vector cells transfected with a plasmid construct. CIA was induced in DBA/1 mice by immunization with native bovine type II collagen (CII). Chinese hamster ovary (CHO) fibroblasts transfected with the mouse IL‐13 or IL‐4 genes were inoculated subcutaneously on days 10 and 25 post‐priming with CII and mice were monitored for signs of arthritis by observers unaware of the status of the animals. Incidence and severity of CIA were significantly reduced in the groups of mice treated with IL‐13 and IL‐4 gene‐transfected CHO cells compared to control groups receiving nontransfected cells. Expression of various cytokines in spleen cells from individual mice was assessed by quantitative reverse transcriptase‐polymerase chain reaction at different times after immunization. Our data show that IL‐13‐induced suppression of CIA coincided with a decreased TNF‐α mRNA expression in the spleen of treated animals. This may explain at least partially the anti‐inflammatory effects of IL‐13 in CIA. Thus, our results may have important implications for the clinical use of T helper (Th)1/Th2 modulatory cytokines as therapeutic agents in the treatment of autoimmune diseases.
Antigenicity of HIV‐derived T helper determinants in the context of carrier recombinant proteins: effect on T helper cell repertoire selection
Authors: Manca Fabrizio , De Berardinis Piergiuseppe , Fenoglio Daniela , Ombra Maria Neve , Li Pira Giuseppina , Saverino Daniele , Autiero Monica , Lozzi Luisa , Bracci Luisa , Guardiola John
Published: March 2007

T helper (Th) epitopes can be included in a recombinant protein with B and CTL epitopes to create more effective immunogens. To determine whether the antigenicity of HIV Th epitopes is preserved in this altered molecular context, human Th clones specific for peptides of HIV gp120 and reverse transcriptase p66 were challenged with recombinant proteins carrying the antigenic epitopes in different sites. We found that a given epitope was recognized by a specific T cell clone only when it was inserted in a particular position of the carrier. However, the permissive position was not the same for all epitopes. Enzymatic excision from a nonpermissive context or insertion of a polyserine spacer between the epitope and the carrier restored antigenicity. Nevertheless, antigenicity was not abolished in a synthetic peptide encompassing the epitope and the neighboring residues from the nonpermissive location. These data suggest that, in this case, the primary sequence of the chimeric protein flanking the HIV peptide is not responsible for loss of antigenicity. Furthermore, constructs carrying the epitope in a given position were recognized by peptide‐specific Th clones raised from some individuals, but not from others. We show that this is due neither to individual modes of processing nor to the use of distinct major histocompatibility complex MHC class II restriction elements, but rather that it is related to the fine specificity of the clones. To study the effect of epitope context on selection of T cell repertoire in a naive individual, T cell lines were generated in vitro by stimulation with different peptide constructs. This resulted in the induction of diverse clonotypes defined by the pattern of recognition of different constructs, by T cell receptor Vβ gene usage and by fine epitope mapping.
Exogenous and endogenous antigens are differentially presented by mast cells to CD4+ T lymphocytes
Authors: Frandji Pierre , Tkaczyk Christine , Oskeritzian Carole , David Bernard , Desaymard Catherine , Mécheri Salaheddine
Published: March 2007

In the present work, we explored the cytokine‐dependent regulation of bone marrow‐derived mast cell (BMMC) antigen‐presenting cell (APC) function, and co‐stimulation requirements, and analyzed the nature of antigens presented to T cells. We observed an up‐regulation of the APC function of mast cells induced by granulocyte/macrophage‐colony‐stimulating factor (GM‐CSF) and a complete abrogation by interferon (IFN)‐γ. Expression of co‐stimulatory molecules CD80 and CD86 was suggested by the ability of mast cells to activate purified lymph node‐derived T cells. Indeed, addition of the fusion protein mCTLA4‐Ig strongly inhibited antigen presentation by mast cells to normal T cells and to the T cell hybridoma 3DO‐54.8. The regulatory mechanisms of APC function by GM‐CSF and IFN‐γ were investigated by measuring CD80 and CD86 transcripts in mast cells. GM‐CSF‐treated mast cells showed a strong increase in the expression of both CD80 and CD86 transcripts, whereas in IFN‐γ‐treated mast cells, this expression was completely abrogated. Thus, up‐ and down‐regulation of CD80 and CD86 expression by GM‐CSF and IFN‐γ is directly correlated to the APC function. In addition, we analyzed antigen presentation by mast cells of endogenous self‐antigens. Mast cells failed to activate anti‐I‐A or anti‐I‐E‐specific T cell hybridomas and alloreactive T cells in primary mixed lymphocyte reactions (MLR). Furthermore, mast cells did not present the mouse β2‐microglobulin (mβ2‐m) peptide 25–40, constitutively expressed on B cells. However, mast cells, especially those treated with GM‐CSF, activated an anti‐mβ2‐m‐specific T cell hybridoma in the presence of exogenous peptide. The minor lymphocyte‐stimulating antigen‐1 Mls‐1a is a viral superantigen (vSAG) encoded by the the mouse mammary tumor provirus‐7 (MMTV‐7). Mast cells, despite a reasonable amount of major histocompatibility complex class II on the cell surface and the presence of MMTV transcripts predicted to encode the vSAG, cannot stimulate in vivo or in vitro Vβ6+ T cells specific for Mls‐1a. In contrast, mast cells could present the exogenous bacterial SAG, staphylococcal enterotoxin B (SEB), to specific Vβ8+ T cells. The selective ability of mast cells to present exogenous antigens may have physiological relevance in that mast cells could participate in immune response regulatory mechanisms by discriminating self from nonself.
Differential nuclear localization of p50, p52, and RelB proteins in human accessory cells of the immune response in situ
Authors: Feuillard Jean , Körner Marie , Israel Alain , Vassy Jeanny , Raphael Martine
Published: March 2007

The Rel/NF‐ϰB proteins, p50, p52, p65, c‐Rel, and RelB, constitute a family of transcription factors involved in the positive regulation of a variety of genes during the immune response. Recently, it has been shown that RelB knockout mice have no dendritic cells (DC). An overexpression of p50 has been described in follicular dendritic cells (FDC). A constitutive NF‐ϰB activity has been reported in mature macrophages. This led to the hypothesis that some of the Rel/NF‐ϰB proteins were key nuclear factors in functions of accessory cells of the immune response. Therefore, we investigated in situ the nuclear localization of Rel/NF‐ϰB proteins in accessory cells of the immune system by immunohistochemistry and double labeling by immunofluorescence from five normal human tonsils and five lymph nodes with follicular hyperplasia. Nuclear p65 and c‐Rel proteins were found in all cell types including lymphocytes. In germinal centers GC, p50, p52, and RelB were found in the nuclei of FDC only and were not detected in the nuclei of CD68+ cells. In T cell areas, p50, p52, and RelB were found in the nuclei of HLA‐DR+ cells with an antigen‐presenting cell (APC) morphology. p52 and RelB were detected in the nuclei in both CDla+ and CD68+ cells from the T cell area, whereas p50 was found only in CD68− and CDla− cells. Cells with nuclear p50 were negative for the CD38, CD20 and CD2 markers. These results show that, physiologically, high levels of nuclear of p50, p52 and RelB are restricted to accessory cells of the immune system, which include FDC in GC, and DC and macrophages in the T cell zone, that specialized scavenger macrophages from GC do not have detectable levels of p52 and RelB, whereas macrophages from the T cell area, known to present the antigen to T cells, do have both nuclear p52 and RelB, and that in the T cell zone, p52 and RelB are located in nuclei of both CDla+, CD68+ or both, cells APC, whereas p50 is restricted to CDla− and CD68− APC. The different patterns of p50, p52 and RelB protein nuclear localization may provide insight into their different roles during the immune response in vivo.
Partial correction of the TH2/TH1 imbalance in neonatal murine responses to vaccine antigens through selective adjuvant effects
Authors: Barrios Christy , Brandt Christian , Berney Monika , Lambert Paul‐Henri , Siegrist Claire‐Anne
Published: March 2007

We have recently shown that neonatal responses to a pannel of vaccine antigens and presentation systems differed qualitatively from adult responses by a bias towards a TH2 pattern. Here we report that a selected adjuvant comprising block copolymers in a water‐in‐oil emulsion can induce balanced TH1/TH2 responses in BALB/c mice primed at 1 week of age with an immunodominant tetanus peptide vaccine. However, using this specific TH1‐driving adjuvant only at time of boosting was not sufficient to fully circumvent the persisting influence of TH2‐biased neonatal responses. Unexpectedly also, a significant local toxicity was observed in newborn and young mice, whereas only mild reactions occurred in adults. Thus, although the induction of strong TH1 responses in the neonatal period can be achieved using specific adjuvants, through modulation of the immunological environment present at time of priming, whether such immunization strategies would be safe in the neonatal period remains to be demonstrated. These observations should be taken into consideration in the development of novel vaccines that will have to be already effective early in life.
Interleukin‐7 induces T cell proliferation in the absence of Erk/MAP kinase activity
Authors: Crawley James B. , Willcocks Joannie , Foxwell Brian M. J.
Published: March 2007

Interleukin (IL)‐7 and IL‐2 are important lymphoproliferative cytokines which both use the γc chain as part of their respective receptors. To learn more of their signaling mechanisms a comparison was made of the patterns of intracellular tyrosine phosphorylated proteins induced by these cytokines in the murine T cell line, CT6. Several similarities were revealed in the tyrosine phosphorylated proteins induced. However, a notable subset of proteins of mainly < 60 kDa were only phosphorylated by IL‐2. Characterization of the two most prominent bands of this subset, pp54 and pp42, revealed these to contain Shc and p42MAP/Erk kinase, respectively. Further studies confirmed that IL‐7 was unable to induce the phosphorylation of either the p44MAP/Erk or p42MAP/Erk or activation of the kinases. Shc is involved in activation of p21ras, a key event in the signaling cascade, via p72raf and MEK, leading to MAP/Erk kinase (MAPK) activation. These data indicate that this pathway is not utilized by IL‐7 and may not, therefore, be essential for cytokine‐driven T cell proliferation. This possibility was supported by studies with the MEK inhibitor PD098059, which had no selective effect on CT6 proliferation induced by IL‐2 as compared with IL‐7, although the drug completely inhibited MAP/Erk phosphorylation induced by IL‐2.
Mouse mammary tumor virus production by thymic epithelial cells in vivo
Authors: Nabarra Bernadette , Desaymard Catherine , Waché Anne‐Claude , Papiernik Martine
Published: March 2007

Exogenous mouse mammary tumor viruses (MMTV) replicate in the mammary glands of infected females, and so infect the suckling pups. We have previously shown that the virus is rapidly disseminated to all the lymphoid organs, including the thymus. The present electron microscope immunohistochemical study describes the viral production site in the thymus. Viral buds and viral proteins were restricted to the thymus medullary epithelial cells. MMTV‐encoded proteins were identified on the free viral particles and on the budding ones, the ribosomes, the membrane of the endoplasmic reticulum, and on the membrane of the medullary type II epithelial cell vacuolar network. The thymus medullary epithelial cells can thus integrate the virus and allow viral replication. The results support earlier results indicating that in some experimental conditions, epithelial cells may be involved in MMTV‐induced negative selection by showing that thymic epithelial cells do express MMTV‐encoded proteins.
Loss of the β1 subunit of the sodium pump during lymphocyte differentiation
Authors: Masat Linda , Cascalho Marilia , Wabl Matthias
Published: March 2007

The Na, K‐ATPase, or sodium pump, is responsible for maintaining cellular volume and is involved in receptor‐mediated endocytosis; it is a ubiquitous transmembrane enzyme in higher eukaryotes and consists of an α and a β subunit. In the mouse, two isotypes of β with no known function have been identified: β1 and β2. We have studied the expression of β1 and β2 in lymphocytes from bone marrow, spleen, peripheral blood, and thymus. The β2 subunit is not expressed in any of the lymphocytes tested. Pre‐B lymphocytes and the majority of mature, resting B cells in the bone marrow express the β1 subunit, as do all pre‐T cells and mature thymocytes. In the spleen and in blood, β1 expression defines subsets of T and B lymphocytes. Mitogen‐stimulated T and B cells lose β1 expression and do not express β2. While there is no indication that there is a change in α subunit isoform expression as a result of lymphocyte activation or that it is expressed in smaller amounts, there is a switch in the expression of the β isoform.
Monovalent single‐chain Fv fragments and bivalent miniantibodies bound to vesicular stomatitis virus protect against lethal infection
Authors: Kalinke Ulrich , Krebber Anke , Krebber Claus , Bucher Etienne , Plückthun Andreas , Zinkernagel Rolf M. , Hengartner Hans
Published: March 2007

Several antibody‐dependent mechanisms have been postulated to mediate neutralization of different animal viruses, including blocking of docking to receptors, induction of conformational changes in the virus coat, and Fc‐dependent opsonization. We have studied the molecular requirements for antibody‐mediated neutralization of vesicular stomatitis virus (VSV) in vitro and protection against lethal disease in vivo with a single‐chain Fv fragment (scFv) and the corresponding bivalent miniantibody (scFv‐dHLX) generated from a VSV‐neutralizing monoclonal antibody. Both monovalent scFv and bivalent scFv‐dHLX miniantibodies were able to neutralize VSV in vitro and to protect interferon‐αβ receptor‐deficient (IFN‐αβR−/−) mice against lethal disease after intravenous injection of 50 plaque‐forming units (pfu) VSV pre‐incubated with the scFv reagents. Similarly, severe‐combined immunodeficient (SCID) mice infected with immune complexes of 108 pfu VSV and bivalent scFv‐dHLX were protected against lethal disease; however, mice infected with immune complexes of 108 pfu VSV and monovalent scFv were not. Although repeated scFv‐dHLX treatment reduced virus quantities in the blood, neither SCID nor IFN‐αβR−/− mice were protected against lethal disease after passive immunization and subsequent VSV infection. This was due to the short half‐life of 17 min of scFv‐dHLX in the circulation. These data demonstrate that neutralization of VSV and protection against lethal disease do not require Fc‐mediated mechanisms and that cross‐linking is not crucial for protection against physiologically relevant virus doses in vivo.
Dual T cell receptor T cells have a decreased sensitivity to physiological ligands due to reduced density of each T cell receptor
Authors: Blichfeldt Erik , Munthe Ludvig André , Røtnes Jan Sigurd , Bogen Bjarne
Published: March 2007

A considerable fraction of T cells express two distinct T cell receptors (TCR), mainly due to expression of two TCR α chains. It has been suggested that such dual‐TCR cells could have a role in autoimmunity. However, as such cells express less of each TCR, they could be less sensitive to their physiological ligand, i.e. peptide plus major histocompatibility complex molecules (MHC). We tested this hypothesis in a transgenic TCR model in which most T cells express different amounts of the transgene‐encoded TCR, due to expression of endogenous TCR α chains. Five Th1 clones derived from λ2315 immunoglobulin light chain‐specific TCR‐transgenic mice expressed different levels of the transgene‐encoded TCR, ranging from ∼ 10000 to ∼ 50000 TCR per cell. Cytosolic Ca2+ mobilization in single T cells from these clones elicited by λ2315 peptide‐pulsed, I‐Ed‐expressing antigen‐presenting cells, correlated linearly with the relative transgene‐encoded TCR expression. The peptide requirement for half‐maximal T cell proliferation showed a similar correlation, with low TCR levels requiring higher peptide concentration. Corroborative evidence was obtained by deployment of short‐term polyclonal CD4+ lines from TCR‐transgenic mice. Such lines had reduced early (Ca2+ mobilization) and late (lymphokine and proliferation) responses, compared with T cell lines from recombination‐deficient TCR‐transgenic severe combined immunodeficiency mice (which express only a single transgene‐encoded TCR). Taken together, the Ca2+ responses increase gradually with increasing TCR expression per cell, similar to the previously described analog Ca2+ signaling elicited by increasing amounts of peptide/MHC [Røtnes et al., Eur. J. Immunol. 1994. 24: 851]. Surprisingly small reductions in TCR expression per cell reduce T cell responsiveness. This suggests that dual‐TCR T cells are immunologically less effective than single‐TCR T cells.
Antigen‐independent responsiveness to interleukin‐4 demonstrates differential regulation of newborn human T cells
Authors: Early Eavan M. , Reen Denis J.
Published: March 2007

The low incidence of graft‐versus‐host disease following clinical use of umbilical cord blood compared to adult bone marrow as a source of stem cells for bone marrow reconstitution, leads to questions concerning the level of immunocom‐petence of newborn T cells. The maturation and functional status of newborn CD4+ T cells, which are almost exclusively CD45RA+ naive T cells, compared with their adult phenotypic counterparts, is poorly understood. We examined the proliferative response to mitogens and cytokines of CD4/CD45RA+ T cells from adults and newborns, with and without accessory cells. Newborn CD4/CD45RA+ T cells demonstrated a distinct proliferative response profile which was determined by the number of accessory cells present in co‐cultures with various stimuli. Newborn CD4/CD45RA+ T cells were particularly responsive to interleukin (IL)‐4, IL‐4 plus anti‐CD2 monoclonal antibodies (mAb) and IL‐4 plus phytohemagglutinin (PHA), whereas adult CD4/CD45RA+ T cells were unresponsive under similar conditions. The mitogenic responses of newborn and adult CD4/CD45RA+ T cells to PHA and anti‐CD2 mAb, which were equivalent, were directly proportional to the number of accessory cells present, whereas the responsiveness to cytokines was inversely proportional to the number of co‐cultured accessory cells. Anti‐CD2 responses were much more sensitive to low numbers of accessory cells than PHA. The particular sensitivity of newborn CD4/CD45RA+ T cells to IL‐4 represents an antigen‐independent T cell activation response which could help promote a Th2 immune response resulting in the newborn.
Thiols prevent Fas (CD95)‐mediated T cell apoptosis by down‐regulating membrane Fas expression
Authors: Delneste Yves , Jeannin Pascale , Sebille Eric , Aubry Jean‐Pierre , Bonnefoy Jean‐Yves
Published: March 2007

Thiol antioxidants have been shown to protect cells against apoptosis. Based on the role of Fas in T cell apoptosis, we investigated the effect of thiols on Fas expression. We report that the thiol N‐acetyl‐L‐cysteine (NAC) prevents the induction of Fas on human peripheral blood T cells in a dose‐dependent manner. It also down‐regulates in a time‐ and dose‐dependent manner Fas expression on Fas‐expressing T cells. Although these effects were not mediated through de novo glutathione synthesis, only compounds with a free thiol group decreased membrane Fas expression. The decrease of Fas expression induced by NAC was not associated with a modulation of Fas mRNA transcription nor with an inter‐nalization, suggesting that NAC may affect the processing of Fas. Indeed, a soluble immunoreactive form of Fas was detected by ELISA and by Western blotting in the supernatants of Fas‐expressing T cells treated with NAC. As a functional consequence, NAC partly protected Jurkat cells against Fas‐mediated apoptotic cell death. Thus, this study shows that, by regulating Fas expression, the cytoprotective properties of NAC can be extended to Fas‐mediated cell death.
Tumor necrosis factor receptor p55 mediates deletion of peripheral cytotoxic T lymphocytes in vivo
Authors: Speiser Daniel E. , Sebzda Eric , Ohteki Toshiaki , Bachmann Martin F. , Pfeffer Klaus , Mak Tak W. , Ohashi Pamela S.
Published: March 2007

Cellular death of activated lymphocytes down‐regulates immune responses and is involved in maintaining self tolerance. Signals associated with ligation of the membrane molecule Fas lead to lymphocyte apoptosis, but additional, Fasindependent mechanisms have been postulated. Here, we show a marked expansion and prolonged persistence of functional activated cytotoxic T cells in mice lacking the tumor necrosis factor (TNF) receptor p55. In the absence of this receptor, peripheral lymphocyte apoptosis was significantly reduced in vivo. The prolonged thymocyte survival was associated with functional anergy, since the T cells no longer proliferated in vitro when stimulated with peptide antigen. However, specific cytotoxic effector function was easily detected in vitro. We conclude that the TNF receptor p55 is involved in peripheral T cell deletion in vivo.
Restricted appearance of self‐reactive clones into T cell receptor intermediate cells in neonatally thymectomized mice with autoimmune disease
Authors: Moroda Tetsuya , Iiai Tsuneo , Kawachi Yasuyuki , Kawamura Toshihiko , Hatakeyama Katsuyoshi , Abo Toru
Published: March 2007

Neonatally thymectomized (NTx) mice fall victim to such autoimmune diseases as gastritis and pancreatitis with aging. Self‐reactive T cell clones are known to be consistently generated through TCR intermediate (i.e. TCRint) cell differentiation in normal mice (i.e. via the extrathymic pathways and an alternative intrathymic pathway). It was investigated whether the generation of such clones in NTx mice follows this rule or whether they are generated by default via mainstream T cell differentiation in the thymus. The majority of T cells generated in NTx mice were TCRint cells in all organs tested. In contrast to athymic mice, which carry only TCRint cells with aging, a leaky appearance of high TCR (i.e. TCRhi) cells emerged in the lymph nodes and other organs of NTx mice. Self‐reactive clones estimated by anti‐Vβ monoclonal antibodies in conjunction with the Mls system were confined to TCRint cells in all tested organs, including a target organ, the stomach, in NTx mice. A leaky population of TCRhi cells did not contain a significant number of self‐reactive clones. Moreover, such self‐reactive clones among TCRint cells in NTx mice with autoimmune disease were shown to be nonanergic in the proliferation assay. The present results suggest that the generation of self‐reactive clones is totally due to TCRint cell differentiation, although it is still undetermined whether the expanding TCRint cell population is generated via the extrathymic pathway or an alternative intrathymic pathway. It is shown here not to be due to a failure of the TCRhi cell‐differentiation pathway in NTx mice.
Genetic control of anti‐idiotypic vaccination against coronavirus infection
Authors: Yu Mathilde W. N. , Lemieux Suzanne , Talbot Pierre J.
Published: March 2007

The idiotypic network can be experimentally altered to induce protective immune responses against microbial pathogens. Both internal image and noninternal image anti‐idiotypic (anti‐Id) antibodies have been shown to trigger antigen (Ag)‐specific immune responses. Therefore, mechanisms of anti‐Id vaccination appear to go beyond structural mimicry of Ag, but remain undefined. Using the neurotropic murine coronavirus animal model, we have previously shown that a polyclonal noninternal image anti‐Id (Ab2) could vaccinate BALB/c mice. To characterize its mode of action, we have examined the immune modulating capability of this Ab2 in vivo in strains of mice with different H‐2 haplotypes. Even though only internal image anti‐Id are expected to induce non‐genetically restricted immunity, this noninternal image Ab2 induced protective immunity in four of eight genetically different strains of mice susceptible to coronavirus infection. These were BALB/c (H‐2d), DBA/1 (H‐2d), DBA/1 (H‐2q), and SWR (H‐2q) mice. Protection was generally correlated with the induction of specific antiviral Ab (Ab3) that showed biological properties, such as virus neutralization in vitro, similar to the initial Ab1. To evaluate the genetic implication of the H‐2 haplotypes in protection, congenic mice were also tested. Vaccination profiles suggest that cooperation between background gene(s) of the BALB/c mouse with H‐2d and H‐2q loci is necessary for an optimal protective immune response, although the main genetic element(s) regulating the antiviral response to Ab2 inoculation appeared to be located outside the major histocompatibility complex. These results are consistent with the ability of Ab2 to induce protective antiviral antibodies in genetically different animals by biological mimicry.
Interferon‐γ rescues HLA class Ia cell surface expression in term villous trophoblast cells by inducing synthesis of TAP proteins
Authors: Rodriguez Anne‐Marie , Mallet Valérie , Lenfant Françoise , Arnaud Jacques , Girr Maryse , Urlinger Stéphanie , Bensussan Armand , Le Bouteiller Philippe
Published: March 2007

Human placental trophoblast cells that constitute the materno‐fetal interface during pregnancy escape maternal alloimmune attack. The different trophoblast cell subpopulations have developed efficient regulatory mechanisms to prevent expression of β2‐microglobulin‐associated HLA class Ia molecules at their cell surface. We previously reported the presence of HLA class Ia messages in villous cytotrophoblast cells and in the syncytiotrophoblast differentiated in vitro purified from term placenta. In this study, we found that these transcripts are translated in heavy chain proteins that are endoglycosidase H sensitive and thus retained in the endoplasmic reticulum or cis‐Golgi. Moreover, these class Ia heavy chains can be co‐immunoprecipitated with the chaperone protein calnexin resident in the endoplasmic reticulum. When these trophoblast cells are treated with interferon (IFN)‐γ, part of the class Ia heavy chains become endoglycosidase H resistant, demonstrating that they have left the endoplasmic reticulum. Furthermore, after such a treatment, these heavy chains are detectable at the cell surface of these trophoblast cells, as assessed by two‐color flow cytometry analysis and immunoprecipitation of cell surface biotinylated proteins using the W6/32 anti‐HLA class I monoclonal antibody (mAb). IFN‐γ treatment induces a significant enhancement of the transcription of transporters associated with antigen processing (TAP1 and TAP2) rather than an increase of HLA class I or β2‐microglobulin messages. Finally, we demonstrate that an anti‐TAP1 mAb coimmunoprecipitates TAP1 proteins and HLA class Ia heavy chains in these IFN‐γ‐treated trophoblast cells. Thus, the constitutive absence of HLA class Ia cell surface expression in term villous cytotrophoblast and syncytiotrophoblast is likely to be due to a lack of transporter proteins that participate in the proper assembly of these molecules in the endoplasmic reticulum. Such a defect can be modified upon IFN‐γ treatment.
Antigen presentation by interferon‐γ‐treated thyroid follicular cells inhibits interleukin‐2 (IL‐2) and supports IL‐4 production by B7‐dependent human T cells
Authors: Lombardi Giovanna , Arnold Kate , Uren Julia , Marelli‐Berg Federica , Hargreaves Roseanna , Imami Nesrina , Weetman Anthony , Lechler Robert
Published: March 2007

The consequence of recognition of antigen on antigen‐presenting cells that are induced to express major histocompatibility complex (MHC) class II molecules following an inflammatory process is still not clear. In this study, we have investigated the outcome of antigen presentation by epithelial cells and we have used as a model thyroid follicular cells (TFC) that are known to express MHC class II molecules in autoimmune thyroid diseases and acquire the capacity to present autoantigens to T cells infiltrating the thyroid gland. The result show that MHC class II‐expressing TFC were unable to stimulate a primary T cell alloresponse, using CD4+ T cells from three HLA‐mismatched responders. Phenotypic analysis showed that TFC, after incubation with interferon‐γ, do not express the co‐stimulatory molecules B7‐1 (CD80) and ‐2 (CD86). Addition of murine DAP.3 cells expressing human B7‐1 (DAP.3‐B7) to cultures containing peripheral blood CD4+ T cells and DR1‐expressing TFC led to a proliferative response, suggesting that the failure of TFC to stimulate a primary alloresponse was due to a lack of co‐stimulation. Similarly, HLA‐DR‐restricted, influenza‐specific T cell clones dependent on B7 for co‐stimulation did not respond to peptide presented by TFC; again the lack of response could be overcome by co‐culture of TFC with DAP.3‐B7. Furthermore, recognition of antigen on TFC inhibited interleukin‐2 (IL‐2) production in the B7‐dependent T cells. In contrast, in T helper type 0 (Th0) T cells, IL‐4 release was not affected by TFC presentation. In addition, antigen presentation by TFC favored IL‐4 production relative to IL‐2 production by B7‐indpendent Th0 clones. These results suggest that antigen presentation by MHC class II+ TFC may induce tolerance in autoreactive Th1 cells but may simultaneously favors a Th2 response in uncommitted T cells, and thereby support autoantibody production.
The cytoplasmic domain of rat NKR‐P1 receptor interacts with the N‐terminal domain of p56lck via cysteine residues
Authors: Campbell Kerry S. , Giorda Roberto
Published: March 2007

NKR‐P1 is a type II transmembrane protein which acts as an activation receptor on natural killer (NK) cells. The cytoplasmic domains of the CD4, CD8 and 4‐1BB receptors contain the sequence Cys‐X‐Cys‐Pro which is directly involved in coupling to another pair of cysteines in the N‐terminal domain of the src family tyrosine kinase p56lck. The cytoplasmic domain of NKR‐P1 in rodents also contains the Cys‐X‐Cys‐Pro sequence, but the capacity of the receptor to bind p56lck is presently unknown. We tested for direct coupling between these proteins using both protein biochemistry and the yeast two‐hybrid technique. Immunoprecipitation studies showed that p56lck can be co‐immunoprecipitated with NKR‐P1 from a rat NK tumor cell line. In addition, the cytoplasmic domain of NKR‐P1 interacted with the N‐terminal domain of p56lck in yeast as assessed by reporter gene activation. Integrity of the cysteine pairs in both proteins was critical in mediating the interaction. The experiments suggest that the association of p56lck with NKR‐P1 is somewhat weaker than the p56lck association with CD8α, but of much lower avidity than between CD4 and p56lck. This could reflect a higher activation threshold for the NKR‐P1 and CD8 receptors, which are involved in cytolytic responses, compared to CD4 which is involved in T cell helper function.
Cyclophosphamide‐induced blood and tissue eosinophilia in contact sensitivity: Mechanism of hapten‐induced eosinophil recruitment into the skin
Authors: Satoh Takahiro , Chen Qui‐Ji , Sasaki Go , Yokozeki Hiroo , Katayama Ichiro , Nishioka Kiyoshi
Published: March 2007

The mechanism leading to selective production and accumulation of eosinophils in certain allergic skin diseases is unknown. Cyclophosphamide treatment (150 mg/kg) of BALB/c mice 48 h before sensitization with picryl chloride (PCI) resulted in striking blood and tissue eosinophilia, maximal at 13 days. Blood eosinophilia was not induced by the sensitization with oxazolone and 2, 4‐dinitrofluorobenzene. Challenge with 1% PCI, but not croton oil caused preferential eosinophil accumulation into the dermis, which was associated with the enhanced expression of vascular cell adhesion molecule 1 (VCAM‐1) on endothelial cells. Intraveneous administration of anti‐VCAM‐1 monoclonal antibody abrogated eosinophil infiltration. In this murine model, we examined the role of several cytokines, including chemokines in inducing selective tissue eosinophilia in vivo. Local administration of antibodies against interleukin (IL)‐1β, IL‐4, tumor necrosis factor (TNF)‐α, and RANTES, but not against IL‐5 before challenge inhibited hapten‐induced eosinophil recruitment. Intradermal injection of recombinant (r)IL‐1β, rIL‐4, rTNF‐α, rRANTES, and rMIP‐1α induced marked eosinophil accumulation. Nonetheless, intradermal rIL‐5 was not a chemoattractant for eosinophils in vivo. Our findings suggest that IL‐1β, IL‐4, TNF‐α, and RANTES contribute to the selective accumulation of eosinophils in contact sensitivity reaction. Although circulating IL‐5 can activate eosinophils and prolong their survival, locally secreted IL‐5 is not crucial for inducing eosinophil recruitment into the skin.
A transgenic T cell receptor restores thymocyte differentiation in interleukin‐7 receptor α chain‐deficient mice
Authors: Crompton Tessa , Outram Susan V. , Buckland Jennifer , Owen Michael J.
Published: March 2007

Interleukin‐7 (IL‐7) receptor α chain‐deficient (IL‐7Rα‐/‐) mice have severely depleted lymphocyte populations and thymocyte development is arrested at the double‐negative (DN) stage. We show that thymocyte development in these mice can be reconstituted by the introduction of a transgenic T cell receptor (TCR), implying that one function of the IL‐7Rα chain is to initiate TCR gene rearrangement. Expression of the recombinase‐activating genes RAG1 and RAG2 was greatly reduced in the IL‐7Rα‐/‐ thymuses, and in DN thymocytes from the TCR transgenic IL‐7Rα‐/‐ mice, but was restored in double‐positive thymocytes from the TCR transgenic IL‐7Rα‐/‐ mice. These data suggest that the IL‐7Rα chain controls RAG expression and initiation of TCRβ chain VDJ rearrangement in DN cells. In contrast, once cells have progressed beyond the DN stage of development the IL‐7Rα chain becomes no longer essential for RAG expression.
X‐SCID B cell responses to interleukin‐4 and interleukin‐13 are mediated by a receptor complex that includes the interleukin‐4 receptor α chain (p140) but not the γ chain
Authors: Matthews David J. , Hibbert Linda , Friedrich Karlheinz , Minty Adrian , Callard Robin E.
Published: March 2007

This study investigates the effect of interleukin (IL)‐4 mutant proteins and a monoclonal antibody to the IL‐4 receptor α chain on IL‐4 and IL‐13 response by B cells from X‐linked severe combined immunodeficiency (X‐SCID) patients in which the common γ chain (γc chain) gene mutations have been fully characterized and no γc chain expression was detected. In this γc chain gene knockout model, it was confirmed that the γc chain is essential for B cell responses to IL‐2 but not for IL‐4 or IL‐13. Dose‐response curves for X‐SCID and normal B cell responses to IL‐4 were indistinguishable, showing that the loss of the γc chain did not diminish the sensitivity of B cells to IL‐4. The mutant protein IL‐4Y124Dand an antibody to the IL‐4R α chain both inhibited responses of X‐SCID B cells to IL‐4 and IL‐13, showing that X‐SCID B cell responses to these cytokines are mediated by a receptor complex that includes the IL‐4R α chain but not the γc chain. Another mutant protein, IL‐4R88D, which has greatly reduced affinity for IL‐4Rα, was found to inhibit responses by normal B cells to IL‐4 but not to IL‐13. IL‐4R88Ddid not, however, inhibit X‐SCID B cell responses to IL‐4. This result is consistent with IL‐4R88Dinhibition of responses mediated by receptor complexes that include the γc chain. We propose that X‐SCID B cells responses to IL‐4 are mediated by an IL‐13 receptor complex comprised of the IL‐4R α chain associated with the recently cloned IL‐13R binding protein. This model has major implications for understanding normal B cell responses to IL‐4.
Direct and sequential switching from μ to ε in patients with Schistosoma mansoni infection and atopic dermatitis
Authors: Baskin Berivan , Islam Khalid B. , Evengård Birgitta , Emtestam Lennart , Smith C. I. Edvard
Published: March 2007

Immunoglobulin isotype switching to IgE in patients infected with Schistosoma mansoni and patients with atopic dermatitis was studied. Patients with parasitic infections or allergic diseases have a higher production of IgE. We found a fourfold increased production of Iε RNA in both patient groups as compared to control donors. The increased expression of germ‐line transcripts correlates with higher serum IgE levels. Nested primer polymerase chain reaction was used to generate Sμ/Sε fragments from DNA of patient peripheral blood mononuclear cells. Twenty‐nine out of fourty sequenced switch fragments had undergone direct joining from Sμ to ε whereas seven fragments showed mono sequential switching from Sμ via either Sμ, Sγ2, Sγ4 or Sε to Sε and four fragments demonstrated double sequential switch: Sμ/Sμ/Sγ1/Sε, Sμ/Sγ2/Sε/Sε or Sμ/Sγ1/Sγ2/Sε. The sequential switching had occurred either via deletions or inversions. Mapping of the breakpoints showed hot spots for recombination within Sμ, Sγ1 and Sε. To our knowledge, this is the first in vivo study in humans demonstrating that switching to IgE can occur from sequential rearrangements via γ1, γ2 or γ4.
Regulation of interleukin‐12 receptor β1 chain expression and interleukin‐12 binding by human peripheral blood mononuclear cells
Authors: Wu Chang‐You , Warrier Rajeev R. , Wang Xin , Presky David H. , Gately Maurice K.
Published: March 2007

The interleukin‐12 receptor (IL‐12R)β1 chain is an essential component of the functional IL‐12R on both human T and natural killer cells. In this report it is shown that activation of human peripheral blood mononuclear cells (PBMC) with anti‐CD3 monoclonal antibody (mAb) or phytohemagglutinin resulted in the up‐regulation of IL‐12Rβ1 expression and IL‐12 binding. Kinetic studies revealed that maximum expression of IL‐12Rβ1 and IL‐12 binding occurred on days 3–4. Anti‐CD3‐induced expression of IL‐12Rβ1 chain and IL‐12 binding by PBMC was augmented by anti‐CD28 mAb, indicating that the potentiating effect of anti‐CD28 on T cell responses to IL‐12 could be mediated, at least in part, by the enhancement of IL‐12R expression. Among 16 cytokines tested, IL‐2, IL‐7 and IL‐15 markedly induced IL‐12Rβ1 expression and IL‐12 binding on resting PBMC, whereas IL‐1α and tumor necrosis factor‐α had a minimal enhancing effect. In contrast, IL‐3, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐12, interferon (IFN)‐α, IFN‐γ, granulocyte/macrophage colony‐stimulating factor and transforming growth factor (TGF)‐β2 had no detectable enhancing effect. Anti‐CD3‐induced expression of IL‐12Rβ1 and of low‐affinity IL‐12 binding sites was partially inhibited by TGF‐β2, IL‐10 and IL‐4; however, TGF‐β2 and IL‐10 completely abolished anti‐CD3‐induced expression of high‐affinity IL‐12 binding sites. Consistent with the reduction of high affinity IL‐12 binding sites, PBMC activated with anti‐CD3 mAb in the presence of TGF‐β2 or IL‐10 failed to produce IFN‐γ or to proliferate in response to IL‐12. These results suggest that Th2 cell‐derived cytokines can inhibit IL‐12‐induced biological functions by inhibiting IL‐12R expression and that expression of a second subunit of the IL‐12R (IL‐12Rβ2), required for the formation of high‐affinity IL‐12 binding sites, may be more highly regulated by TGF‐β2 and IL‐10 than is expression of IL‐12Rβ1.