bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection from experimental autoimmune diabetes in the non‐obese diabetic mouse with soluble interleukin‐1 receptor
Authors: Nicoletti Ferdinando , di Marcou Roberto , Barcelliniu Wilma , Magro Gaetano , Schorlemmeru Hans U. , Kurrleu Roland , Lunettau Michele , Grasso Sebastiano , Zacconeu Paola , Meronif Pierluigi
Published: March 2007

We have evaluated the effects of a treatment with soluble interleukin‐1 receptor (sIL‐1R) in the accelerated model of autoimmune diabetes induced by cyclophosphamide (CY) in the non‐obese diabetic (NOD) mouse. Prior to the CY challenge (350 mg/kg body weight), female euglycemic NOD mice were randomly divided into three groups (A–C). Groups B and C were treated daily from 1 day before to 13 days after the CY challenge with sIL‐1R at doses of 0.2 and 2 mg/kg body weight. Group A was treated with PBS. By 2 weeks after CY administration, an acute form of autoimmune diabetes with glycosuria, hyperglycemia and severe insulitis occurred in the majority (13/20, 65%) of the control mice (group A). In contrast, repeated injections with sIL‐1R protected NOD mice from insulin‐dependent diabetes mellitus (IDDM) development in a dose‐dependent fashion; the incidence of IDDM was 53.3% (8/15) in the mice treated with 0.2 mg/kg and only 6.7% (1/15) in those treated with 2 mg/kg. However, none of the doses of the sIL‐1R reduced the extent of insulitis in NOD mice. Importantly, the anti‐diabetogenic property of sIL‐1R may not involve major T cell function impairment; accordingly, in parallel experiments, splenic lymphoid cells from NOD mice not challenged with CY, but treated with 2 mg/kg sIL‐1R for 5 consecutive days showed a normal distribution of mononuclear cell subsets and maintained their capacity to secrete interferon‐γ and IL‐2 and to proliferate in response to polyclonal mitogenic stimulation with concanavalin A.
Developmental shift in the patterns of interleukin production in early post‐natal life
Authors: Coutinho Graça Cunha , Delassusu Sylvie , Kourilskyu Philippe , Bandeira António , Coutinho António
Published: March 2007

In mice, immunological tolerance to self is established in the perinatal period, when tolerance susceptibility to allogenic tissues is higher than in adults. We have now investigated whether this could result from developmental regulation of effector functions of T cells exposed to specific antigens, by studying the “natural” or T cell receptor‐induced expression of several interleukin genes. We used qualitative and quantitative polymerase chain reaction methods to study interleukin (IL)‐2, IL‐4, IL‐10 and interferon‐γ mRNA expression by splenic cells at different ages. The results show that newborn peripheral cells (up to day 7), in contrast to the T lymphocytes of adult mice, express high levels of IL‐4 and interferon‐γ, and very low levels of IL‐2 messenger spontaneously and upon specific T cell activation. This characteristic phenotype depends on intrinsic T cell properties, as it is not due to the newborn environment.
Presentation of viral antigens restricted by H‐2Kb, Db or Kd in proteasome subunit LMP2‐and LMP7‐deficient cells
Authors: Zhou Xianzheng , Momburgu Frank , Liuu Tianmin , Motalu Ussama M. Abdel , Jondalu Mikael , Hämmerlingu Günter J. , Ljunggren Hans‐Gustaf
Published: March 2007

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I‐mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane‐spanning ATP‐binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA‐2 by HLA‐A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H‐2Kb, Db or Kd genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus‐specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H‐2Kb, Db or Kd class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.
Lymphotoxin acts as an autocrine growth factor for Epstein‐Barr virus‐transformed B cells and differentiated Burkitt lymphoma cell lines
Authors: Gibbons Deena L. , Roweu Martin , Cope Andrew P. , Feldmann Marc , Brennan Fionula M.
Published: March 2007

A critical event in B cell immortilization by Epstein‐Barr virus (EBV) is the establishment of an autocrine loop where cells produce a growth factor which supports their own proliferation. We investigated the potential of lymphoblastoid cell lines (LCL) and Burkitt lymphoma (BL) cell lines to produce and respond to the cytotoxins, tumor necrosis factor‐α (TNF‐α) and lymphotoxin (LT). Transformation in vitro of peripheral blood B cells by EBV from seven different donors resulted in spontaneous production of both LT (11542 pg/ml ± 7546, mean ± SD) and, to a lesser extent, TNF‐α (197 pg/ml ± 174). Similarly BL cell lines derived from in vivo transformation which developed a ‘LCL‐like’ phenotype in vitro (group III) produced more LT (1990 pg/ml ± 1740) than the ‘group I’ BL cell lines (< 40 pg/ml LT) which had maintained the original BL biopsy cell phenotype in vitro. Transformation of periphreal blood B cells to generate LCL also resulted in an increase in surface p75 (p < 0.02) and to a lesser extent p55 (not significant, ns) TNF receptor (TNF‐R) expression. Similar increases in surface TNF‐R (p75 p < 0.02, p55 ns) were observed on the ‘group III’ BL cell lines compared with the ‘group I’ BL cell lines. Proliferation of an LCL and a ‘group III’ BL cell line in vitro was via an autocrine loop since inhibition of LT reduced proliferation. This proliferation could also be blocked in the presence of the antagonistic anti‐p55 TNF‐R antibody, H398, but not the antagonistic antibody anti‐p75 TNF‐R antibody UTR‐1. Furthermore, proliferation could be induced with the p55 agonistic antibody, HTR‐9. In contrast to these observations with p55 TNF‐R antibodies, two out of six of the ‘group III’ BL lines (Jijoye and Oba) only expressed the p75 TNF‐R and proliferation of these cells could only be blocked by the antagonistic anti‐p75 TNF‐R antibody UTR‐1. These data suggest that LT is an autocrine growth factor for lymphoblastoid cells, and BL cell lines which display an LCL phenotype. Furthermore, although both TNF‐R are increased on the surface of these cells, this autocrine growth signal is mediated principally through binding to the p55 TNF‐R.
Ineffective expression of CD40 ligand on cord blood T cells may contribute to poor immunoglobulin production in the newborn
Authors: Brugnoni Duilio , Airò Paolo , Gralfu Daniel , Marconiu Massimo , Lebowitzu Morena , Plebaniu Alessandro , Gilianiu Silvia , Malacarne Fabio , Cattaneo Roberto , Ugaziou Alberto G. , Albertiniu Alberto , Kroczeku Richard A. , Notarangelo Luigi D.
Published: March 2007

A major feature of the immature immune system in the newborn is its inability to produce significant levels of immunoglobulins other than IgM in response to antigens. It has recently been demonstrated that interaction of the CD40 molecule on B cells with the CD40 ligand (CD40L) on activated T cells is pivotal for immunoglobulin switching. In view of these findings, we have tested cord blood mononuclear cells (CBMC) for expression of CD40L. Our data clearly demonstrate that freshly isolated CBMC do not express significant levels of CD40L upon in vitro activation; this defect is intrinsic to CD4− cord blood lymphocytes. In vitro priming of CBMC with phytohemagglutinin and interleu‐kin‐2 for several days induced a conversion from the “naive” to the “memory” phenotype (as assessed by expression of CD45 isoforms) and led to substantial CD40L expression upon appropriate restimulation. These data indicate that ineffective CD40L expression might represent a major factor for reduced immunoglobulin production in the neonate, and suggest that antigenic exposure in vivo leads to changes in the genetic program, enabling T cells to express CD40L.
CD14 is expressed and functional in human B cells
Authors: Ziegler‐Heitbrock H. W. Löms , Pechumer Heinrich , Petersmannu Irmhild , Durieuxu Jean‐Jacques , Vitau Natalio , Labetau Mario O. , Ströbelf Marion
Published: March 2007

The human B cell line RPMI 8226 exhibits variable staining with the CD14 antibody My4. We have isolated three stable clones from this line with clones 1 and 2 being My4 positive and clone 3 My4 negative. Similar to previous results in monocytes, immunoprecipitation with the My4 antibody revealed a 54‐kDa cell surface molecule, analysis of supernatants showed soluble CD14, and Northern blotting demonstrated a 1.4‐kb transcript in clones 1 and 2, but not in clone 3, which suggests that the My4 antibody detects CD14 in clones 1 and 2. This CD14 molecule was functional in that lipopolysaccharide stimulation induced interleukin (IL)‐6 and IL‐10 in clones 1 and 2 but not in clone 3. Furthermore, the My4 antibody was capable of blocking these responses at the transcript and protein levels. Finally, peripheral blood B cells were highly purified by cell sorting (> 98% CD19 positive). These cells produced IL‐6 in response to lipopolysaccharide, and this response was blocked by anti‐CD14 antiserum. Thus, our findings demonstrated that human B cells can express functionally active CD14.
Wortmannin, an inhibitor of phospholipase D activation, selectively blocks major histocompatibility complex class II‐restricted antigen presentation
Authors: Carrasco‐Marín Eugenio , Alvarez‐Domínguez Carmen , Leyva‐Cobián Francisco
Published: March 2007

Wortmannin, a fungal metabolite, is a specific inhibitor of phospholipase D (PLD) activation. Presentation of defined exogenous soluble proteins to specific T cell hybridomas was studied by using different antigen‐presenting cells (APC): IA‐positive peritoneal macrophages (MΦϕ), B lymphoma cells (B) or dendritic cells (DC). Major histocompatibility complex class II‐restricted antigen presentation by MΦϕ was blocked when cells were pretreated with wortmannin. However, when cells constitutively expressing IA molecules (B. DC) were used as APC, no inhibition was observed. Additionally, MHC class I antigen presentation was not impaired by wortmannin. Moreover, wortmannin does not block either peptide presentation or presentation to autoreactive T cells. This effect was time and dose dependent and occurred at the level of intracellular handling of the antigen. Mainly because it was not a toxic inhibition, it was reversible with time and neither antigen uptake and catabolism, nor IA synthesis were affected. Because MΦ, but not B or DC, express PLD activity and only the former were blocked by wortmannin in antigen presentation, our results strongly suggest that a differential antigen‐processing pathway exists in these disparate APC, which could be based essentially on a wortmannin‐sensitive, PLD‐dependent step present in MΦ but absent and/or unnecessary in both B lymphoma cells and DC.
A 150‐kDa molecule of macrophage membrane stimulates interleukin‐2 and interferon‐γ production and proliferation of ovalbumin‐specific CD4+ T cells
Authors: Agrewala Javed N. , Vinay Dass S. , Joshi Aneesh , Mishra Gyan C.
Published: March 2007

In the present study, we describe the potential co‐stimulatory role of a macrophage membrane‐associated protein of 150 kDa (M150). The protein was isolated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and was found to be a single molecule on two‐dimensional gel electrophoresis. The molecule was re‐constituted in phosphatidyl choline vesicles and tested for its ability to promote the proliferation and the secretion of lymphokines from T helper (Th) cells. The reconstituted M150 induced a significant proliferation of anti‐CD3 monoclonal antibody (mAb)‐stimulated ovalbumin‐specific CD4+ T cells. Further, Th cells activated with this molecule in the presence of anti‐CD3 mAb mainly secreted interleukin (IL)‐2 and interferon‐γ but not IL‐4. M150 could not promote the proliferation of Th cells, or lymphokine secretion in the absence of anti‐CD3 mAb. These observations suggest that M150 acts by selectively activating a Th1‐like immune response.
Ly‐49‐independent inhibition of natural killer cell‐mediated cytotoxicity by a soluble major histocompatibility complex class I molecule
Authors: Roth Claude , Kourilsky Philippe , Ojcius David M.
Published: March 2007

Natural killer (NK) cells lyse their targets in a non‐major histocompatibility complex (MHC)‐restricted manner, and the cytotoxicity can be inhibited by a number of MHC class I allele products, suggesting that NK cells may have a “positive receptor” that recognizes the target and a “negative receptort” that receives an inhibitory signal from class I. Since negative receptors could also exert their effect by masking a positive ligand, we have determined whether there may be a direct interaction between class I and an NK surface receptor by measuring cytotoxicity in the presence of a soluble class I molecule, Kd. Soluble Kd at micromolar concentrations could efficiently block NK cell cytotoxicity, suggesting that class I has a direct effect on cytotoxicity, rather than masking another target cell ligand. Inhibition required that Kd be at least divalent, probably because of its higher affinity or its ability to cross‐link the NK surface receptor. In addition, the effect was independent of the peptide used to load Kd, and there was inhibition of cytotoxicity of NK cells derived from either H‐2d or H‐2b mice. Finally, depletion of NK cells expressing Ly‐49 had no effect on the specific inhibition by Kd, raising the possibility that NK cells are endowed with additional negative receptors besides Ly‐49. Taken together, these results suggest that there may be a family of NK receptors recognizing different class I alleles, which can receive negative signals by directly binding to class I on the target cell surface.
An intracisternal A‐particle sequence codes for an antigen recognized by syngeneic cytolytic T lymphocytes on a mouse spontaneous leukemia
Authors: De Bergeyck Vinciane , De Plaen Etienne , Chomez Patrick , Boon Thierry , van Pel Aline
Published: March 2007

Cytolytic T lymphocyte (CTL) clones directed against spontaneous mouse leukemia LEC have been obtained. By transfecting a cosmid library into cells which were then tested for their ability to stimulate the CTL, we identified the gene coding for the antigen recognized by one of these CTL clones. It is the gag gene of an endogenous defective retrovirus that belongs to the intracisternal A particle (IAP) family. A gag‐encoded nonapeptide presented by the H‐2 Dk molecule caused recognition by the anti‐LEC CTL clone. Southern blot and polymerase chain reaction analyses indicated that the expression of the antigen by the LEC tumor cell line resulted from the transposition of an IAP sequence into a new genomic location.
Response of interleukin‐6‐deficient mice to tumor necrosis factor‐induced metabolic changes and lethality
Authors: Libert Claude , Takahashi Nozomi , Cauwels Anje , Brouckaert Peter , Bluethmann Horst , Fiers Walter
Published: March 2007

Whether interleukin (IL)‐6 contributes to tumor necrosis factor (TNF)‐induced lethal shock or whether, on the contrary, it is part of a protective feedback system, remains unresolved. Here, we report experiments with IL‐6 gene‐disrupted mice (IL‐60/0). We have tested the susceptibility of these to TNF‐induced metabolic changes and lethality in different models, and compared the results with those obtained with IL‐6+/+ wild‐type mice. We studied the response to TNF in three different models: (i) murine TNF administration; (ii) TNF in galactosamine (GalN)‐sensitized mice; (iii) TNF in Bacillus Calmette‐Guérin‐sensitized mice. We observed no significant difference between the two types of mice in any of the three models. Furthermore, IL‐60/0 mice could be equally well desensitized (by IL‐1) to TNF/GalN‐induced lethality and tolerized to TNF‐induced shock as IL‐6+/+ mice. We also observed that, in response to turpentine, TNF or IL‐1, IL‐60/0 mice produced significantly less acute phase proteins (APP) than IL‐6+/+ mice. In IL‐60/0 mice, less corticosterone was induced by TNF than in the control mice, while the response to adrenocorticotropic hormone was the same. The results indicate that IL‐6 is not contributing in a major way to the pathogenesis leading to TNF‐induced shock, and that neither IL‐6 nor the APP studied are essential for a protective feedback system.
Recombinant vaccinia viruses expressing interleukin‐5 stimulate an earlier appearance of antibody‐secreting cells in the lung
Authors: Janardhana Vijaya , Andrew Marion , Thomas Sandra , Coupar Barbara
Published: March 2007

Interleukin‐5 (IL‐5) is a cytokine that participates in the regulation of antibody secretion, in particular promoting the secretion of IgA at mucosal sites. In this report, recombinant vaccinia viruses expressing IL‐5 have been inoculated into mice and the appearance of antibody‐secreting cells in the spleen and lungs investigated. Although vaccinia virus‐expressed IL‐5 did not increase the level of IgA in serum, antibody‐secreting cells, measured in an enzyme‐linked immunosorbent spot assay, appeared earlier in lungs when the immunizing virus expressed IL‐5. These early B cells secreted either IgM or IgG1.
Certain anti‐CD34 monoclonal antibodies induce homotypic adhesion of leukemic cell lines in a CD18‐dependent and a CD18‐independent way
Authors: Traoré Yves , Hirn Joelle
Published: March 2007

CD34 is a highly glycosylated type I membrane protein expressed by early hematopoietic progenitor cells as well as by endothelial cells and a subset of bone marrow stromal cells. CD34 is thought to play an important role during early hematopoiesis, although its function is unknown. We demonstrate that triggering of CD34 results in a rapid and vigorous homotypic adhesion in CD34+ cell lines, thereby providing evidence for a cell‐cell adhesion function of CD34. The cellular adhesion event, induced by only two anti‐CD34 mAb, (Immu‐133 and QBend‐10) was dependent on metabolic energy, an intact cytoskeleton and the presence of divalent cations. Analysis of antibody inhibition experiments indicated that the aggregation process partially involved the CD18 molecule.
B and T lymphocyte subsets enter peripheral lymph nodes and Peyer's patches without preference in vivo: no correlation occurs between their localization in different types of high endothelial venules and the expression of CD44, VLA‐4, LFA‐1, ICAM‐1, CD2 or L‐selectin
Authors: Westermann Jürgen , Nagahori Yutaka , Walter Sebastian , Heerwagen Catharina , Miyasaka Masayuki , Pabst Reinhard
Published: March 2007

Many lymphocytes enter tissues such as peripheral lymph nodes and Peyer's patches through high endothelial venules (HEV). It is known that HEV differ in the expression of adhesion molecules as lymphocyte subsets do. Through the interaction of these molecules B and T lymphocyte subsets are thought to be preferentially directed into lymphoid organs. However, it is unclear which role these mechanisms play in vivo, since there are no studies demonstrating that blood lymphocyte subsets preferentially interact with different types of HEV in vivo. Therefore, in the present study the frequency of B, T, CD4+ and CD8+ lymphocytes in the wall of the HEV of rat peripheral lymph nodes and Peyer's patches was analyzed by immunohistology. In addition, the expression of CD44, VLA‐4, LFA‐1, ICAM‐1, CD2 and L‐selectin on B and T lymphocyte subsets of the blood was determined by flow cytometry. Although B and T lymphocytes showed significantly different levels of expression for each adhesion molecule investigated, the relation of B and T lymphocytes within the HEV of peripheral lymph nodes and Peyer's patches was strikingly comparable (38.0 ± 5.2% vs. 40.6 ± 5.7% and 62.0 ± 5.2% vs. 59.4 ± 5.7%, respectively). The same was true for CD4+ and CD8+ cells. Thus, although HEV and the blood lymphocyte subsets differ markedly in their expression pattern of adhesion molecules, the existing levels are sufficient to mediate comparable entrance of B and T lymphocyte subsets into both types of HEV.
Thymic selection and peptide‐induced activation of T cell receptor‐transgenic CD8 T cells in interleukin‐2‐deficient mice
Authors: Krämer Susanne , Mamalaki Clio , Horak Ivan , Schimpl Anneliese , Kioussis Dimitris , Hünig Thomas
Published: March 2007

The requirement for interleukin‐2 (IL‐2) in repertoire selection and peripheral activation of CD8 T cells was tested in mice rendered IL‐2 deficient by gene targeting and expressing a transgenic T cell receptor (TcR) (F5) specific for influenza nucleoprotein (NP) 366‐374 + H‐2Db. Positive selection of the transgenic F5 TcR into the CD8 compartment proceeded normally. Both in vivo and in vitro, the antigenic peptide induced depletion of immature thymocytes and proliferation of mature CD8 T cells regardless of the presence of an intact IL‐2 gene. In contrast, cytotoxic T lymphocyte (CTL) activity was only generated by T cells from IL‐2+ F5 transgenic mice. Exogenous IL‐2 was able to fully restore the CTL response of IL‐2−/− responder cells in vitro. Thus, both in vivo and in vitro, clonal expansion of CD8 T cells can proceed in the absence of IL‐2, whereas in peptide‐immunized F5 transgenic mice, induction of cytotoxic effector function is IL‐2 dependent.
Changes in thymic export of γδ and αβ T cells during fetal and postnatal development
Authors: Witherden Deborah A. , Kimpton Wayne G. , Abernethy Nevin J. , Cahill Ross N. P.
Published: March 2007

The thymus plays an essential role in the generation and selection of T cells and exports approximately 0.5–1% of thymocytes per day in young animals and considerably fewer in older animals. To date there have been no studies directly examining fetal thymic export in any species. Using the technique of intrathymic injection of fluorescein isothiocyanate, followed by an assay for green fluorescent cells in the periphery and for the expression of cell surface antigens on these cells, we have compared directly the export of T cells from the fetal and postnatal ovine thymus. While the thymus exports both αβ and γδ T cells, our results demonstrate that the proportion of thymic γδ T cells that are exported per day is much higher than that of thymic αβ T cells. Moreover, the export rate of γδ T cells increased from approximately 1 in every 60 γδ thymocytes per day emigrating from the fetal thymus to 1 in every 20 from the postnatal thymus. In addition, we identify a population of CD5+CD4−CD8−γδ− T cells emigrating from the fetal thymus but greatly reduced among thymic emigrants after birth. These findings have several implications regarding the mechanisms and control of selection of both γδ and αβ T cells.
In vivo modification of major histocompatibility complex class II DRA promoter occupancy mediated by the AIR‐1 trans‐activator
Authors: Rigaud Gildas , Paiola Fiorenza , Accolla Roberto S.
Published: March 2007

RJ 2.2.5 is a human B cell mutant derived from the Burkitt lymphoma Raji cell, which is defective in the AIR‐1 locus function. This locus encodes a transcriptional trans‐activator required for the constitutive expression of major histocompatibility complex (MHC) class II genes. Here we show, by in vivo DNase I footprinting, that the AIR‐1 locus defect correlates with changes in the DRA promoter occupancy. Interestingly, reexpression of human MHC class II genes in RJ 2.2.5 × mouse spleen cell hybrids is associated with partial reversion of DRA promoter occupancy to the Raji pattern. DRA promoter occupancy in other class II‐negative B cell lines, derived from patients with bare lymphocyte syndrome, is drastically different from the one observed in RJ 2.2.5 and Raji cells. Moreover, the use of the DNase I as an in vivo footprinting agent reveals that the patients' cell lines do not display a completely “bare promoter” as previously reported using dimethyl sulfate as the footprinting agent. Thus, the use of DNase I allowed us, for the first time, to correlate the AIR‐1 locus defect with class II promoter occupancy alterations and distinguish these alterations from the ones observed in phenotypically similar but genetically distinct MHC class II‐negative cells.
Localization of the site of the murine IgG1 molecule that is involved in binding to the murine intestinal Fc receptor
Authors: Kim Jin‐Kyoo , Tsen May‐Fang , Ghetie Victor , Ward E. Sally
Published: March 2007

Site‐directed mutagenesis of a recombinant Fc hinge fragment has recently been used to localize the site of the murine IgG1 molecule that is involved in the control of catabolism (the “catabolic site”). In the current study, the effects of these CH2 and CH3 domain mutations (Ile 253 to Ala 253, His 310 to Ala 310, Gln 311 to Asn 311, His 433 to Ala 433 and Asn 434 to G1n 434) on intestinal transfer of Fc hinge fragments in neonatal mice have been analyzed. Studies using direct transfer and competition assays demonstrate that the mutations affect the transmission from intestinal lumen into serum in a way that correlates closely with the effects of the mutations on pharmacokinetics. Binding studies of several of the Fc hinge fragments to isolated neonatal brush borders have been used to confirm the in vivo transmission data. These analyses have resulted in the localization of the binding site for the intestinal transfer receptor, FcRn, to specific residues of the murine Fc hinge fragment. These residues are located at the CH2‐CH3 domain interface and overlap with both the catabolic site and staphylococcal protein A (SpA) binding site. The pH dependence of IgG1 or Fc fragment binding to FcRn is consistent with the localization of the FcRn interaction site to a region of the Fc that encompasses two histidine residues (His 310 and His 433). To assess whether one or two FcRn binding sites per Fc hinge are required for intestinal transfer, a hybrid Fc hinge fragment comprising a heterodimer of one Fc hinge with the wild‐type IgG1 sequence and a mutant Fc hinge with a defective catabolic site (mutated at His 310, G1n 311, His 433 and Asn 434) has been analyzed in direct and competition transmission assays. The studies demonstrate that the Fc hybrid is transferred with significantly reduced efficiency compared to the wild type Fc hinge homodimer and indicate that the binding to FcRn, and possibly subsequent transfer, is enhanced by the presence of two FcRn binding sites per Fc hinge fragment.
The efficiency of cysteine‐mediated intracellular retention determines the differential fate of secretory IgA and IgM in B and plasma cells
Authors: Guenzi Silvia , Fra Annamaria , Sparvoli Antonella , Bet Paola , Rocco Mattia , Sitia Roberto
Published: March 2007

Previous studies on IgM secretion demonstrated a role for the μ chain C‐terminal cysteine (Cys575) in preventing the transport of unpolymerized subunits along the secretory pathway. The sequence homology between the C‐terminal tailpieces of μ and α heavy chains prompted us to investigate the role of cysteine‐mediated retention in the control of IgA secretion during B cell development. Similar to IgM, IgA are not secreted by B lymphocytes: the retention mechanism can be reversed by the reducing agent 2‐mercaptoethanol, suggesting that disulfide interchange reactions are involved in the quality control of both IgM and IgA. Yet, α2L2 subunits, but not μ2L2, are secreted constitutively by plasma cells. We demonstrate that the differential retention of IgM and IgA subunits by myeloma transfectants is mainly due to the presence of an acidic residue upstream the α chain C‐terminal cysteine. The regulation of polymeric Ig secretion during B cell development provides an example of how thiol‐mediated quality control can be modulated according to the aminoacidic context surrounding the critical cysteine and to the cell type.
Role of antigen‐presenting cells in the polarized development of helper T cell subsets: evidence for differential cytokine production by Th0 cells in response to antigen presentation by B cells and macrophages
Authors: Duncan David D. , Swain Susan L.
Published: March 2007

Immune challenges can elicit polarized responses skewed towards the development of T helper type 1 (Th1) or Th2 T cell subsets. To determine if distinct antigen‐presenting cells (APC) populations might selectively influence Th subset development, we studied the role of two key APC populations, B cells and macrophages, in the differentiation of effector Th populations from naive precursor Th in vitro. Antigen (Ag)‐specific, naive CD4+ T cells were enriched from a mouse strain, AND, bearing a transgenic α/β T cell receptor (TCR) encoding reactivity with pigeon cytochrome c peptide 88‐104. Peptide Ag was used throughout these studies so that differences in the uptake and processing by the two APC populations would not influence the results. Both APC populations, activated B cells and bone marrow‐derived macrophages, supported the development of effector Th having the capacity to secrete high levels of cytokines when restimulated. Regardless of APC population present during effector development, exogenous interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) had dominant effects on Th subset development. Thus, with both APC populations, effector Th generated in the presence of IFN‐γ acquired a Th1‐type cytokine profile, Th generated with IL‐4 acquired a Th2‐type cytokine profile, and Th generated without IFN‐γ or IL‐4 acquired a Th0‐type cytokine profile. B cells and macrophages also had equivalent APC function in the restimulation of Th1 and Th2‐like effectors, since only minor differences in cytokine production were noted for these effector populations when restimulated with the two APC populations. However, in 8 of 19 experiments, the Th0‐like effector population generated in the presence of IL‐2 differentially responded to restimulation with B cells and macrophages, secreting significantly more IFN‐γ when restimulated with B cells, and significantly more IL‐4 when restimulated with macrophages. We also found that Th effector populations recultured in IFN‐γ or IL‐4 assumed a more Th1 or Th2‐like phenotype, respectively, regardless of their initial cytokine profile. We conclude that through a subtle capacity to skew cytokine production by a Th0 subset, different APC may selectively influence Th subset development under conditions of prolonged or chronic stimulation in an autocrine fashion.
Genetic requirements for acceleration of diabetes in non‐obese diabetic mice expressing interleukin‐2 in islet β‐cells
Authors: Allison Janette , McClive Peter , Oxbrow Leonie , Baxter Alan , Morahan Grant , Miller Jacques F. A. P.
Published: March 2007

Diabetes was dramatically accelerated in non‐obese diabetic (NOD) transgenic mice that expressed interleukin‐2 (IL‐2) in their β cells. A single cross to C57BL/6 completely prevented this effect and a further backcross to the NOD genetic background showed that at least two diabetes susceptibility loci (Idd1s and Idd3/10s) were required for the diabetes acceleration. T cells activated to islet antigens were not circulating in the mice. The accelerating effect of IL‐2 was present, but decreased, in NOD mice that lacked CD8+ T cells as well as in NOD SCID mice. The implications are that in the NOD genetic background, the production of cytokines, such as IL‐2, by islet‐specific CD4+ T cells can lead to β cell damage and diabetes and that CD8+ T cells may have a role in accelerating diabetes onset.
Staurosporine restores signaling and inhibits interleukin‐8‐induced chemotactic desensitization
Authors: Johnston James A. , Ferris Douglas K. , Wang Ji Ming , Longo Dan L. , Oppenheim Joost J. , Kelvin David J.
Published: March 2007

Interleukin 8 (IL‐8) is a chemotactic cytokine (chemokine) that plays a key role in the accumulation and activation of neutrophils at inflammatory sites. In this report we demonstrate that homologous chemotactic desensitization occurs upon pretreatment of neutrophils with IL‐8 or N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and results in the inhibition of neutrophil chemotaxis upon subsequent challenge with the same ligand. This homologous chemotactic desensitization could be prevented by pretreating the neutrophils with the protein kinase inhibitor staurosporine, indicating that protein kinases may play an essential role. The attenuation of homologous desensitization by staurosporine restored chemotaxis but was not associated with a change in IL‐8 receptor expression, affinity or the rate of ligand internalization, indicating that homologous desensitization does not alter ligand‐receptor interaction.

Using two‐dimensional analysis we have shown that IL‐8 induced a rapid serine/threonine phosphorylation of a number of neutrophil substrates the most prominent being phosphoprotein 39 (pp39), extracellular signal‐related kinase‐1, pp55 and pp66. Prior desensitization of neutrophils with IL‐8 blocked all subsequent phosphorylation upon rechallenge with IL‐8. However, the desensitization was specific for IL‐8 since normal phosphorylation of identical substrates was observed in response to FMLP. When neutrophils were pretreated with staurosporine, prior to desensitization, phosphorylation of pp39 was observed upon restimulation with IL‐8. Further study revealed that pp55 and pp66 were not phosphorylated in the presence of staurosporine.

Thus, homologous desensitization of neutrophils in response to IL‐8 does not result from changes in receptor expression, but rather from a staurosporine‐sensitive inactivation of subsequent signal transduction. This desensitization is selective since the cells are able to respond to other ligands.
In vitro priming of cytotoxic T lymphocytes against poorly immunogenic epitopes by engineered antigen‐presenting cells
Authors: Bellone Matteo , Iezzi Giandomenica , Manfredi Angelo A. , Protti Maria Pia , Dellabona Paolo , Casorati Giulia , Rugarli Claudio
Published: March 2007

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides presented by major histocompatibility complex class I (MHC‐I) molecules on the surface of target cells. Optimal induction of CD8+ CTL depends on the amount of relevant peptide/MHC‐I complexes and the presence of co‐stimulatory molecules on antigen‐presenting cells (APC). The antigen‐processing defective mutant cell line RMA‐S, when cultured at low temperature, expresses high amounts of MHC‐I molecules that do not contain endogenously derived peptides. These “empty” MHC‐I molecules can be stabilized by addition of MHC‐binding peptides. RMA‐S cultured at low temperatures with selected peptides have been used for in vitro CTL induction with conflicting results. RMA‐S cells do not express detectable amounts of B7 co‐stimulatory molecule. This could explain their unpredictable efficiency as APC. We have evaluated whether RMA‐S cells, stably transfected with cDNA encoding for the human B7.1 molecule could provide effective co‐stimulation for CD8+ T lymphocytes. RMA‐S/B7 cells, loaded with different synthetic peptides, demonstrated a high and sometimes unique efficiency for in vitro primary CTL induction, even when “sub‐optimal” antigen peptides were used. Most importantly, RMA‐S/B7 cells pulsed with naturally processed peptides extracted from the poorly immunogenic B16 melanoma cells were able to prime CD8+ cells against B16 melanoma. We conclude that the use of RMA‐S/B7 cells as APC represents an ideal strategy for in vitro CTL immunization without prior in vivo priming. This system may also help to address the issue of the different contributions of co‐stimulation and relative occupancy of MHC‐I by single peptide epitopes in CTL priming.
Different kinetic patterns of cytokine gene expression in vivo in orally tolerant mice
Authors: Fishman‐Lobell Jacqueline , Friedman Aharon , Weiner Howard L.
Published: March 2007

A biologically relevant mechanism of generating peripheral tolerance in T cells that have escaped thymic deletion is by the oral administration of soluble antigens. Oral tolerance occurs by two distinct mechanisms. Feeding a single high dose of antigen induces anergy of antigen‐specific TH1 cells, while multiple low doses of antigen induce regulatory Tcells that mediate suppression by producing immunosuppressive cytokines. Although cytokine production in orally tolerant animals has been well studied utilizing in vitro assay systems, semi‐quantitative characterization of cytokine production in oral tolerance in vivo has not been carried out. In this paper we have developed a system using semi‐quantitative RNA polymerase chain reaction to characterize cytokine gene expression in vivo in mice orally tolerized by feeding either a single high dose or multiple low dosages of antigen. We find that measurable differences in interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ) gene expression occurred between the tolerized and non‐tolerized groups. Qualitatively, IL‐4 mRNA was increased in both orally tolerized groups. However, significant differences in IL‐4 gene expression between the two groups in both magnitude and kinetics were found. A large but short‐lived increase in IL‐4 was produced in mice fed a single high dose, while a relatively more moderate, longer‐lived increase was produced in mice fed multiple low doses. The increase in IL‐4 gene expression was specific only to the draining lymph node following antigen administration. Expression of IFN‐γ was decreased in both orally tolerant groups. These results indicate that tolerance in TH1 cells is induced by both of these feeding regimens while TH2 responses are intact and amplified upon reexposure to antigen.
Localization of two cytotoxic T lymphocyte epitopes and three anchoring residues on a single nonameric peptide that binds to H‐2Ld and is recognized by cytotoxic T lymphocytes against mouse tumor P815
Authors: Van den Eynde Benoǐt , Mazarguil Honoré , Lethé Bernard , Laval Françoise , Gairin Jean Edouard
Published: March 2007

Mouse mastocytoma P815 expresses tumor antigens P815A and P815B encoded by a single gene called P1A and carried by a single peptide named P1A 35–43 (NH2‐Leu‐Pro‐Tyr‐Leu‐Gly‐Trp‐Leu‐Val‐Phe‐COOH). P1A 35–43 is presented to anti‐P815A and anti‐P815B cytotoxic T lymphocytes (CTL) by major histo‐compatibility complex (MHC) H‐2Ld molecules. In order to determine the individual role played by each amino acid residue of P1A 35‐43 in binding to H‐2Ld and in recognition by anti‐A and anti‐B T cell receptors (TcR), a series of P1A 35‐43 peptides substituted by alanine at single positions was synthesized and tested for binding to H‐2Ld and for CTL recognition. Binding to H‐2Ld was estimated by measuring the ability of the peptide to up‐regulate cell surface expression of H‐2Ld. We found that three residues were important for interaction of P1A 35‐43 with H‐2Ld. Two of them, Pro at position 2 and Phe at position 9 were consistent with the described H‐2Ld binding motif. A third residue, Trp at position 6, was also required for effective MHC binding of the tumor antigen. CTL sensitization assays showed that alanine substitution at position 7 (Leu) or at position 8 (Val) dramatically affected peptide recognition by anti‐A CTL while positions 3 (Tyr) and 4 (Leu) were critical for recognition by anti‐B CTL. We conclude that Pro2, Trp6 and Phe9 constitute the anchor residues of P1A 35‐43 to H‐2Ld, whereas the dipeptidyl sequences Tyr3‐Leu4 and Leu7‐Val8 form the core epitopes recognized by the TcR of anti‐P815B and anti‐P815A CTL, respectively.
The expansion of a CD4+ T cell population bearing a distinctive β chain in MRL lpr/lpr mice suggests a role for the fas protein in peripheral T cell selection
Authors: Musette Philippe , Pannetier Christophe , Gachelin Gabriel , Kourilsky Philippe
Published: March 2007

MRL lpr/lpr mice suffer from a systemic lupus erythematosus‐like autoimmune disease. The lpr mutation impairs the normal transcription of the fas message, the product of which mediates apoptosis and presumably the proper selection of T cells. We have found an early expansion of CD4+ T cells bearing a distinctive Vβ8.3‐Dβ1.1‐Jβ1.1 T cell receptor β chain in the periphery of MRL lpr/lpr mice, which was not detected in MRL +/+ mice nor in the thymus of MRL lpr/lpr mice. Thus, since thymic selection is normal in MRL lpr/lpr mice, we propose that the lpr mutation results in defective negative selection at the periphery.
Expression of E‐cadherin by murine dendritic cells: E‐cadherin as a dendritic cell differentiation antigen characteristic of epidermal Langerhans cells and related cells
Authors: Borkowski Teresa A. , Van Dyke Byron J. , Schwarzenberger Kathryn , McFarland Vivian W. , Farr Andrew G. , Udey Mark C.
Published: March 2007

Murine epidermal Langerhans cells (LC) synthesize and express E‐cadherin, a homophilic adhesion molecule that mediates adhesion of LC to keratinocytes in vitro. To determine if E‐cadherin expression is characteristic of LC or is a feature of all dendritic cells (DC), we studied DC from various lymphoid tissues and peripheral blood for reactivity with anti‐E‐cadherin monoclonal antibody. By flow cytometry, DC prepared from skin‐associated lymph nodes (LN) expressed E‐cadherin, whereas DC prepared from gut‐associated LN and spleen did not. However, direct comparison revealed that levels of E‐cadherin expressed by DC from skin‐associated LN were ∼fivefold lower than those expressed by freshly‐prepared LC. Immunohistochemical studies confirmed that E‐cadherin was expressed by DC in skin‐associated LN in situ, and demonstrated that the number of E‐cadherin+ DC in LN draining skin previously treated with the contact allergen 2,4,6‐trinitrochlorobenzene was increased relative to the number of E‐cadherin+ DC present in LN draining normal skin. DC propagated from the blood of cyclophosphamide‐treated mice in granulocyte/macrophage‐colony stimulating factor‐supplemented media also expressed E‐cadherin. E‐cadherin immunoprecipitated from DC co‐migrated in SDS polyacrylamide gels with that from fibroblasts transfected with murine E‐cadherin cDNA, and mRNA encoding extracellular and intracellular regions of E‐cadherin was present in DC propagated from blood. These results indicate that E‐cadherin expressed by murine dendritic cells is identical to E‐cadherin expressed by epithelial cells, and suggest that E‐cadherin represents a DC differentiation antigen characteristic of LC and lineage‐related cells (skin‐associated LN DC).
Deficient expression of co‐stimulatory molecules on Leishmania‐infected macrophages
Authors: Kaye Paul M. , Rogers Nicola J. , Curry Allison J. , Scott Julia C.
Published: March 2007

Co‐stimulatory signals are necessary for the full activation of T cells for growth and effector function. As co‐stimulatory molecules are normally regulated in their expression, it has been suggested that microorganisms enhance their expression on host antigen‐presenting cells (APC), thus allowing efficient generation of anti‐microbial immunity. We here describe experiments which demonstrate that infection of macrophages, both in vitro and in vivo, by the protozoan parasite Leishmania donovani fails to trigger expression of co‐stimulatory molecules B7‐1 and heat‐stable antigen on these APC. Furthermore, infection with this parasite inhibits the macrophage response to normal regulatory signals, such as bacterial lipopolysaccharide. These changes in the cell surface are mirrored in functional studies of co‐stimulation in vitro. Together, these data suggest a further facet of parasite interference in host immunity, but also indicate a potential new target for immunotherapy.
Mutation of aspartate 82 of the human C5a receptor abolishes the secretory response to human C5a in transfected rat basophilic leukemia cells
Authors: Monk Peter N. , Pease James E. , Marland Gillian , Barker Michael D.
Published: March 2007

C5a is a potent chemoattractant for monocytes, neutrophils and other leukocytes. The receptor for human C5a is a member of the rhodopsin superfamily of G protein‐coupled receptors and contains an aspartate residue (Asp82) within the putative second transmembrane domain conserved in all other G protein‐linked receptors. We investigated the role of this residue and also the carboxy‐terminal 23 residues of the C5a receptor in ligand binding and signal transduction by expressing wild‐type and mutant receptors in the rat basophilic leukemia cell line RBL‐2H3. Wild‐type and truncated receptors coupled efficiently to effector systems, resulting in the C5a‐dependent discharge of granule contents. In contrast RBL cells transfected with receptors in which Asp82 had been mutated to asparagine did not respond to human C5a by secretion despite binding human C5a with high affinity. We conclude therefore that Asp82 is not involved in the interaction with ligand but is essential for the proper transduction of the ligand binding signal.
Immunolocalization of intracellular interleukin‐4 in normal human peripheral blood basophils
Authors: Mueller Regula , Heusser Christoph H. , Rihs Silvia , Brunner Thomas , Bullock Gillian R. , Dahinden Clemens A.
Published: March 2007

The question as to whether other cell types apart from helper T lymphocytes are capable of producing interleukin‐4 (IL‐4) has gained much interest over the last years. Recent studies indicate that human basophils also produce IL‐4, although direct proof is missing so far. In this study we demonstrate the presence of IL‐4 in the cytoplasm of in vitro activated human peripheral blood basophils derived from normal donors. Cytokine‐producing cells were revealed at the single‐cell level by intracellular immunofluorescence staining using IL‐4‐specific monoclonal antibodies. Basophils showed a characteristic, apparently granular staining pattern easily discerned from the eccentric dot‐shaped staining pattern in activated T cells used in control experiments. Cell counts following priming with IL‐3 and stimulation with polyclonal sheep anti‐IgE antibody or the anaphylatoxin C5a revealed a significant increase in IL‐4‐positive basophils to about 19% as compared with unprimed, unstimulated control cells (6%). The amount of IL‐4 in the supernatant of these cell preparations paralleled these observations with an at least five‐ to sevenfold increase following stimulation as compared with control cells (< 5 ng/ml). Using confocal scanning laser microscopy, the intracellular presence of IL‐4 was confirmed, and the cells were identified as being basophils on terms of their characteristic multilobed nucleus. This observation was supported by double labeling studies using antibodies to IL‐4 and to the high‐affinity IgE receptor (FcεR1). Interestingly, stimulation of cells led to a decrease in the number of FcεR1‐positive cells. The above results show direct evidence that IL‐4 is produced by activated human basophils.