bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell specialization at environmental interfaces: T cells from the lung and the female genital tract of lpr and gld mice differ from their splenic and lymph node counterparts
Authors: Ibraghimov Alexander R. , Lynch Richard G.
Published: March 2007

Mice homozygous for lpr and gld accumulate CD4−CD8− (double‐negative, DN) B220+CD510Thy‐110 αβ T cells in the spleen and lymph nodes (LN), while mucosal gut T cells are normal. To study other mucosa‐associated T cell populations, we examined T cell subsets separated according to expression of αβ T cell receptor, CD4, CD5, CD8, Thy‐1 and B220 in the lung and the female genital tract (FGT) of adult MRL lpr, C3H lpr and C3H gld mice. αβ T cell accumulation was detected in both the FGT and the lungs of lpr and gld mice but, in contrast to the spleen and LN, equal proportions of DN B220+ and CD4+ of CD8+ (single‐positive, SP) B220− T cells were observed in these sites, and the T cells had an increased expression of Thy‐1 and CD5. Staining for CD44, L‐selectin, and CD45RB revealed a higher percentage of effector/memory T cells in lpr and gld lungs and FGT compared to spleens and LN. CD69 expression suggested chronic activation of DN and SP T cells in lpr and gld lungs and FGT. Thus, we show that FGT and lung resident T cells are affected by lpr and gld mutations, but that their phenotypes are distinct from those of systemic T cells. These data suggest that T cells associated with FGT and lung mucosal tissues represent a separate lineage from systemic T cells, and/or that the abnormal T cells in lpr and gld mice are selected against in mucosal surfaces exposed to environmental antigen.
Analysis of the cytolytic activity mediated by natural killer cells from acquired immunodeficiency syndrome patients in response to phytohemagglutinin or anti‐CD16 monoclonal antibody
Authors: Sirianni Maria Caterina , Mezzaromau Ivano , Aiutiu Fernando , Morettaf Alessandro
Published: March 2007

The aim of this study was to assess the cytolytic potential of natural killer (NK) cells from human immunodeficiency virus type 1 (HIV‐1)‐infected patients, at different stages of the disease. Twenty HIV‐1 seronegative donors as well as sixty HIV‐1 seropositive patients were studied. Phytohemagglutinin and/or the anti‐CD16 monoclonal antibody Kd1 were used to redirect the peripheral blood lymphocyte lysis of these patients to the 51Cr‐labeled Fcγ receptor‐positive P815 murine mastocytoma target cell line. In parallel, NK cytotoxicity to tumor targets was investigated. Seronegative as well as HIV‐1 Center for Disease Control (CDC) stage II patients showed maintained cytolytic activity. The cytolytic potential declined with disease progression, starting with CDC IVC2 patients, and was strongly diminished in acquired immunodeficiency syndrome stage patients. This defect was accompanied by decreased cytolytic activity to tumor targets and was not corrected by the in vitro addition of interleukin‐2. The number of cells bearing a mature NK phenotype was normal in all the study groups. Our data suggest that the impaired NK cytotoxicity to tumor targets described during the progression of HIV‐1 disease may be related to the progressive loss of function of surface receptors involved in NK cell triggering.
Exogenous superantigens acutely trigger distinct levels of peripheral T cell tolerance/immunosuppression: Dose‐response relationship
Authors: Miethke Thomas , Wahl Claudia , Gaus Helmut , Heeg Klaus , Wagner Hermann
Published: March 2007

Ligand‐specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. T cell receptor (TcR) transgenic mice offer a clear advantage for studying post‐thymic tolerance mechanisms in vivo that are operational in a monoclonal T cell population with preselected antigen specificity. Yet it is unclear whether the rules defined in monoclonal T cells of genetically manipulated mice reflect those operative in clonally diverse peripheral T cells of normal mice. To analyze acute tolerance mechanisms in unselected peripheral T cells, we challenged normal mice with the superantigen staphylococcal enterotoxin B (SEB) and analyzed ligand‐reactive Vβ8+ T cells for TcR‐triggered tolerance mechanisms such as anergy, TcR down‐regulation, or apoptosis. Upon challenge with graded doses of SEB (0.001–10 μg) Vβ8− T cells become anergic within 6–16 h. Importantly, a dosage effect of SEB in regard to the level of anergy induced was observed. Anergy induced by low concentrations of SEB (0.001–0.1 μg) is transient and is overcome by clonal growth, while higher concentrations of SEB (0.1–10 μg) cause long‐lasting anergy resistant to cell cycle progression. At high SEB concentrations (1–10 mg) about 50% of the anergic Vβ8+ T cells additionally down‐regulate their TcR‐CD3 complex, followed by a loss of CD2, CD4, CD8 accessory molecules. In parallel, T cell phenotypenegative but genotypically Vβ8+ T cells are generated. The T cell phenotypenegative cells reacquire their Vβ8+ T cell phenotype upon culture in vitro. In vivo, a subset of Vβ8+ cells, defined by an intermediate stage of TcR down‐regulation, i.e. Vβ8lowCD3+ cells, but not T cell phenotype‐negative cells are selectively programmed for apoptosis, which occurs within 1 h. These data suggest that SEB triggers distinct tolerance pathways which operate in a hierarchical fashion in clonally diverse ligand‐reactive T cells. Specifically, the results illustrate the power of exogenous superantigens to exploit these distinct tolerance pathways, thereby achieving distinct levels of immunosuppression.
Double‐negative thymocyte subsets in CD3′ chain‐deficient mice: Absence of HSA+CD44−CD25− cells
Authors: Crompton Tessa , Mooreu Marilyn , MacDonald H. Robson , Malissen Bernard
Published: March 2007

Double‐negative (DN) thymocyte subsets were examined in mice deficient in the CD3′ chain (ζ −/−). The HSA +CD44−CD25− subset was found to be missing, and DN thymocytes seemed to differentiate directly from HSA+CD25+CD44−cells to double‐positive (DP) cells. When fetal thymic ontogeny was examined, we found a marked difference between ζ −/− embryos and heterozygous littermates from embryonic day 17.5, in terms of CD25, CD4 and CD8 expression, and thymus size. The ζ −/− thymocytes failed to down‐regulate CD25 and to expand exponentially. The cell cycle status of adult thymocyte subsets indicated that although the HSA +CD25−CD44− subset was missing, the CD25+ DN population contained normal numbers of cycling cells, and the CD25+ DP cells (which were not detectable in normal mice) contained 5–10% cells in G2/M + S. Taken together these data suggest that the CD3′ chain might have a specific role in the control of proliferation of DN thymocytes during T cell development. Our data clearly show that one can dissociate the signal for a CD25+ DN cell to differentiate (which occurs in the absence of CD3′), from a signal to proliferate and from loss of cell surface CD25.
Neuropeptides are potent modulators of human in vitro immunoglobulin E synthesis
Authors: Aebischer Iwan , Stämpfli Martin R. , Zürcher Adrian , Miescher Sylvia , Urwyler Adrian , Freyu Brigitte , Lugeru Thomas , Whiteu Richard R. , Stadler Beda M.
Published: March 2007

We determined the effect of adrenocorticotropin hormone (ACTH) on the regulation of IgE synthesis. Depending on the concentration, ACTH enhanced or inhibited IgE synthesis in a culture system where IgE synthesis was induced with interleukin‐4 (IL‐4) and anti‐CD40 monoclonal antibody in peripheral blood mononuclear cells. Similar effects on IgE synthesis were observed by adding ACTH‐related peptides, e.g. corticotropin‐releasing factor (CRF), the inducer of ACTH, or α‐melanocyte stimulating hormone (α‐MSH), a cleavage product of ACTH. However, ACTH had no effect on IgG or IgM synthesis in this culture system. ACTH did not act directly on either B or T cells as there was no influence on IgE synthesis in a system using purified B cells alone or co‐cultured with T cells. The effect of ACTH on IgE synthesis was mediated by accessory cells. This was shown by priming purified CD14‐positive monocytes with ACTH and reconstitution experiments. Therefore, these findings suggest that ACTH and the related peptides CRF and α‐MSH can influence the microenvironment modulating an IL‐4 and anti‐CD40 monoclonal antibody driven class switching to IgE via accessory cells.
Restricted T cell receptor usage in DA rats during early collagen‐induced arthritis
Authors: Erlandsson Helena , Müsseneru Åsa , Klareskogu Lars , Goldf Daniel P.
Published: March 2007

We have investigated the usage of T cell receptor (TcR) Vβ gene structures in DA rats undergoing homologous collagen‐induced arthritis induced by immunization with homologous collagen type II emulsified in Freund's incomplete adjuvant. TcR Vβ expression within freshly isolated inflamed synovial tissue (ST) and primary draining lymph nodes was analyzed in a semi‐quantitative polymerase chain reaction assay. A highly restricted TcR Vβ gene expression was observed in ST samples 2 days after onset of clinical disease, with Vβ6, 8.2 and 8.5 comprising more than 60% of the total Vβ expression measured. The corresponding primary draining lymph nodes from the diseased animals showed some bias in Vβ expression at this time but not as remarkable as that found in ST. One week after onset of disease, consistent Vβ gene bias was much less evident in either site. These results indicate that T cells which infiltrate synovia early in disease are highly restricted with respect to Vβ expression.
A human CD4 transgene rescues CD4−CD8+ cells in β2‐microglobulin‐deficient mice
Authors: Baron Agnès , Hafen Katrin , von Boehmer Harald
Published: March 2007

The specificity of the αβ T cell receptor for class I or class II major histocompatibility complex (MHC) molecules determines whether a mature T cell will be of the CD4−CD8+ or CD4+CD8− phenotype, respectively. We show here that a human CD4 transgene can rescue a significant fraction of CD4−CD8+ T cells in β2‐microglobulin‐deficient mice. Cells with this phenotype could be induced to become potent killers of targets expressing allogeneic MHC antigens, indicating that lineage commitment can precede the rescue of developing cells by the T cell receptor for antigen and the CD4 coreceptor.
Defective lymphokine production by most CD8+ and CD4+ tumor‐specific T cell clones derived from human melanoma‐infiltrating lymphocytes in response to autologous tumor cells in vitro
Authors: Guilloux Yannick , Viret Christophe , Gervois Nadine , Dréan Eric Le , Pandolfino Marie‐Christine , Diez Elisabeth , Jotereau Francine
Published: March 2007

Human melanomas are infiltrated by tumor‐reactive T lymphocytes. However, the ability of these cells to elicit a specific anti‐tumor response in vivo remains to be established. Because lymphokine production is critical for T cell functions, we have analyzed the capacity of melanoma‐specific tumor‐infiltrating lymphocyte (TIL) clones to produce major lymphokines: interleukin‐2 (IL‐2), interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4), as well as tumor necrosis factor (TNF), in response to direct antigen presentation by autologous and allogeneic tumor cells. We report here that, upon stimulation by autologous melanoma cells, all TIL clones secreted TNF but only a few of them produced significant amounts of IL‐2, IL‐4 or IFN‐γ. Nonetheless, all these clones consistently produced two or three of these last lymphokines upon stimulation with phorbol myristate acetate and calcium ionophore, as well as IL‐2 upon CD3 stimulation, showing the existence of three lymphokine profiles among them: Th1, Th0 and a profile characterized by IL‐2 and IL‐4, but not IFN‐γ secretion. Stimulation of TIL clones by allogeneic melanoma lines sharing the appropriate HLA‐peptide complexes revealed that defective IL‐2 production seemed to be a constant feature for some clones, while it was, for other clones, dependent on the antigen‐presenting tumor cells. For this last type of clone, we further showed that defective IL‐2 induction resulted from an LFA‐3 defect of some melanoma cells or from distinct yet undefined defects of other melanoma lines. Our data suggest that defective lymphokine secretion may be an essential component of the in vivo failure of melanoma‐reactive TIL to control tumor development. Interestingly both CD4+ and CD8+ TIL clones from one patient were fully activated by the autologous melanoma cells in vitro, supporting a potential role of such TIL in spontaneous or induced tumor rejection.
Evidence for a selective and multi‐step model of T cell differentiation: CD4+CD8low thymocytes selected by a transgenic T cell receptor on major histocompatibility complex class I molecules
Authors: Pircher Hanspeter , Ohashi Pamela S. , Boyd Richard L. , Hengartner Hans , Brduscha Karin
Published: March 2007

We have characterized a prominent (15‐20 %) thymocyte population expressing CD4 at a high and CD8 at a low level “CD4+8lo” in mice transgenic for a T cell receptor “TCR” restricted by major histocompatibility complex “MHC” class I molecules. The results demonstrate that the CD4+8lo population is an intermediate stage between immature CD4+8+ and end‐stage CD4+8‐ thymocytes and that the survival of these cells crucially depends on the successful interaction of the transgenic TCR with self MHC class I molecules. In addition we demonstrate that the avidity of the interaction between TCR and self MHC class I molecules determines whether CD4+8lo thymocytes are found in significant numbers in this transgenic model. Our findings support a selective and multi‐step model of T cell differentiation in the thymus.
Signaling properties of anti‐immunoglobulin — resistant variants of WEHI‐231 B lymphoma cells
Authors: Benhamou Laure E. , Watanabe Takeshi , Kitamura Daisuke , Cazenave Pierre‐André , Sarthou Pierre
Published: March 2007

Stimulation of the B cell antigen receptor (BCR) of the murine immature WEHI‐231 B lymphoma with anti‐immunoglobulin antibodies leads to irreversible growth arrest and apoptosis. As in normal B cells, membrane immunoglobulin (mIg) ligation in WEHI‐231 cells triggers a series of signaling cascades from the BCR to intracellular compartments. In order to address the role of early signals in mediating the growth arrest of WEHI‐231 cells, we have generated two variants resistant to the anti‐Ig‐mediated inhibitory effect. Some of the properties of these variants have been recently described in terms of bcl‐2 and c‐myc gene regulation. We report here that these variants can be further distinguished from the wild type on the basis of significant alterations in the early biochemical events which follow mIg ligation. Both Ca2+ signals and patterns of protein tyrosine phosphorylation were affected in these variants, suggesting that alterations in the early signal transduction machinery may have profound effects on the fate of B cells. In addition, we found that expression of the p75HSI substrate of p53/56lyn was strikingly reduced in both variants as compared to the wild type. These findings support the view that p75HSI may play a critical role in BCR‐dependent signaling cascades.
Promiscuous T cell recognition of an H‐2 IA‐presented mycobacterial epitope
Authors: Vordermeier H. Martin , Harris David P. , Moreno Carlos , Ivanyi Juraj
Published: March 2007

Genetically permissive T cell epitopes are an important prerequisite for the development of peptide‐based vaccines or immunodiagnostic reagents. We have investigated the structural requirements of permissive T cell recognition of peptide p350—369 from the 38‐kDa antigen of Mycobacterium tuberculosis. This peptide was found to be immunogenic in mice of the H‐2b, bm12, d. s and k, but not of the H‐2f genotype. T cell responses were restricted by I‐A class II molecules. The same epitope core was recognized in the H‐2b, d and k genotypes. T cell hybrids from BALB/c and C57BL/10 mice were used to determine: (i) the critical residues using substituted peptide derivatives and (ii) the degree of T cell promiscuity. Two out of five BALB (H‐2d)‐derived hybridomas tested displayed promiscuous peptide recognition in the context of H‐2b and H‐2bm12 antigen‐presenting cells. The recognition of critical residues was found to be uniform for all five hybridomas when tested with syngeneic antigen‐presenting cells; additional critical residues were identified when the peptide was recognized in the context of allogeneic antigen‐presenting cells. Only one of the four tested C57BL/10 (H‐2b) hybridomas showed promiscuity in the context of H‐2bm12. Each of these C57BL/10‐derived clones had a distinct response profile toward the critical residues. We propose that the demonstrated T cell promiscuity involves peptide interaction with polymorphic H‐2 I‐A residues.
Injection of detergent‐denatured ovalbumin primes murine class I‐restricted cytotoxic T cells in vivo
Authors: Schibeck Reinhold , Böhm Waltraud , Reimann Jörg
Published: March 2007

Complex adjuvant formulations have been used to introduce soluble protein antigens into the “endogenous” processing pathway and hence to elicit specific, major histocompatibility complex class I‐restricted cytotoxic T lymphocyte (CTL) responses. We tested if simple modifications of a model protein antigen, i.e. ovalbumin (OVA), can render it immunogenic for murine class I‐restricted CTL when injected into mice in soluble form. Injection of 1—100 μg native OVA into C57BL/6 (H‐2b) mice did not stimulate a class I‐restricted CTL response. In contrast, immunization of mice with 0.5 to 10 μg sodium dodecyl sulfate (SDS)‐or deoxycholate (DOC)‐denatured OVA efficiently primed CD8+ CTL specific for the well‐characterized Kb‐restricted OVA257—264 epitope. Gel‐purified SDS‐denatured OVA devoid of protein fragments and excess detergent efficiently stimulated a specific CTL response in vivo. OVA preparations denatured by heat or urea treatment were not immunogenic for murine CTL. Injection of non‐treated or detergent‐treated, antigenic OVA257—264 peptide into mice did not elicit a CTL response. Thus, denaturation of OVA by simple detergents such as SDS or DOC dramatically enhances its immunogenicity for class I‐restricted CTL but not all modes of denaturation are equally effective.
Human granzyme B is essential for DNA fragmentation of susceptible target cells
Authors: Su Bin , Bochan Markian R. , Hanna William L. , Froelich Christopher J. , Brahmi Zacharie
Published: March 2007

We have partially characterized the granules of the human NK cell line, YT‐INDY, and assessed granule‐mediated lysis and DNA fragmentation of assorted targets. Biochemical studies demonstrated significant quantities of granzyme B (asp‐ase) and a heretofore undescribed chymase but no tryptase (i.e., granzyme A or 3) or distinct met‐ase. YT‐INDY expressed mRNA for granzyme B, perforin and CCPX. The existence of perforin was confirmed by immunoblot. The granules lysed both human and murine NK‐sensitive and NK‐resistant targets. YTindependent lytic pathway is associated with the granules. In addition, 4‐(2‐aminoethyl) benzenesulfonylfluoride hydrochloride (AEBSF), an inhibitor that selectively blocked the chymase and 3,4‐dichloroisocoumarin (DCI), an inhibitor that inactivated both chymase and asp‐ase activities, marginally affected lysis. By gel electrophoresis and 125I‐labeled deoxyuridine release assay, only murine cells (SP2/0 and YAC‐1) underwent DNA fragmentation, and cleavage was completely inhibited by DCI, whereas EGTA, AEBSF and aurintricarboxylic acid (ATA) had no effect. The results, therefore, underscore the central role of granzyme B in granule‐mediated DNA fragmentation, emphasize that the protease acts via an ATA‐resistant endonuclease pathway and stress that nucleolysis does not invariably accompany granule‐mediated cytolysis. Finally, ATA inhibited the asp‐ase activity of isolated but not granule‐associated granzyme B. ATA, therefore, is not a specific endonuclease inhibitor and results obtained with ATA should be viewed cautiously.
Different cytokine production profiles of γδ T cell clones: Relation to inflammatory arthritis
Authors: Chomarat Pascale , Kjeldsen‐Kragh Jens , Quayle Alison J. , Natvig Jacob B. , Miossec Pierre
Published: March 2007

This study was performed to investigate whether γδ T cells could also be divided into subsets, identified by a cytokine profile, as described for αβ T helper (Th) cell subsets. Cytokine production was studied in 22 γδ T cell clones obtained from the synovial fluid and peripheral blood of one patient with inflammatory arthritis and compared to that of 26 αβ T cell clones of the same and different patients. Interferon‐γ (IFN‐γ) was produced by 18 (82%) and interleukin‐4 (IL‐4) by 17 (77%) out of 22 γδ T cell clones, respectively. In contrast, IL‐10 was not produced, except at very low level in one case. The mean levels of IL‐4 were lower for clones derived from synovial fluid. When considering the production of IFN‐γ as an indicator of Th1 and that of IL‐4 as an indicator of Th2, respectively, the most common pattern was a γδTh1‐like pattern, with the combination of high levels of IFN‐γ and low levels of IL‐4. This pattern was found in Vδ1+ clones, all from synovial fluid. Additional patterns were also observed: a mixed, probably γδ Th0‐like pattern with a more balanced production of both IFN‐γ and IL‐4; a γδTh1 pattern with the production of IFN‐γ alone; a γδTh2 pattern with the production of IL‐4 alone. These three patterns were also seen in blood γδ T cells which were all Vδ2, indicating that these patterns were independent of the γδ phenotype. γδ T cell clones produced lower levels of IFN‐γ (p = 0.001) and higher levels of IL‐4 than αβ clones (p < 0.02). These differences in cytokine production between αβ and γδ subsets and within these subsets may contribute to their respective role in chronic inflammation.
Antigen‐driven tissue‐specific suppression following oral tolerance: Orally administered myelin basic protein suppresses proteolipid protein‐induced experimental autoimmune encephalomyelitis in the SJL mouse
Authors: Al‐Sabbagh Ahmad , Miller Ariel , Santos Leonilda M. B. , Weiner Howard L.
Published: March 2007

Immunomodulatory treatment paradigms have been applied to animal models of T cell‐mediated autoimmune diseases in an attempt to develop an immunospecific and non‐toxic form of therapy which can be applied to humans. These treatment paradigms are often directed to T cells with a restricted T cell receptor repertoire or that react with dominant peptide determinants. Experimental data, however, suggests that even if the initial T cell response is restricted to a specific self‐protein in the target organ, spreading autoimmunity may develop with broadening of T cell autoreactivity to additional epitopes of the same autoantigen or to different autoantigens in the target organ. Thus, multiple autoantigens may become targets of the autoimmune response. This makes immunotherapeutic strategies based on suppressing responses to restricted proteins or clones of cells problematic. We have previously shown that suppression of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat by oral myelin basic protein (MBP) is mediated by the release of transforming growth factor‐β after triggering by the oral tolerogen. Here, we report that in the SJL model of EAE oral administration of an autoantigen from the target tissue suppresses disease independent of whether it is or is not the inciting antigen. Thus, orally administered MBP or MBP peptides suppress proteolipid protein (PLP)‐induced EAE, whereas intravenously administered MBP does not. Both oral and intravenous PLP, however, suppressed PLP disease. These findings have important implications for the use of oral tolerance as a therapeutic approach for the treatment of T cell‐mediated inflammatory autoimmune diseases in man in which the inciting autoantigen is unknown or in which there is autoreactivity to multiple autoantigens in the target tissue.
Major histocompatibility complex class I allele‐specific peptide libraries: Identification of peptides that mimic an H‐Y T cell epitope
Authors: Gavin Marc A. , Dere Beverley , Grandea Andres G. , Hogquist Kristin A. , Bevan Michael J.
Published: March 2007

We describe a novel method for screening large libraries of random peptides for T cell antigens. Two libraries were constructed, containing fixed amino acids representing the major histocompatibility complex (MHC) class I anchor residues for H‐2Kb‐restricted octamers and H‐2Db‐restricted nonamers. Peptides from the Kb‐restricted library (KbL: SXIXFXXL) and the Db‐restricted library (DbL: XXXXNXXXIM) specifically stabilize empty Kb and Db molecules, respectively. The libraries contain peptides that mimic several H‐2b‐restricted cytotoxic T lymphocyte epitopes, and 21 mimotopes for a Db‐restricted H‐Y epitope were isolated. A degenerate synthetic peptide of limited complexity containing the identified H‐Y sequence motif was found to be similar to the natural H‐Y epitope by reverse‐phase high performance liquid chromatography analysis. This peptide is also capable of immunizing female mice against male splenocytes. Several applications for MHC‐restricted peptide libraries are discussed.
Selective inhibition of E‐selectin, ICAM‐1, and VCAM in endothelial cells
Authors: Stuhlmeier Karl M. , Csizmadia Vilmos , Cheng Qi , Winkler Hans , Bach Fritz H.
Published: March 2007

Endothelial cells, as they normally exist in the vasculature as quiescent cells, perform several functions. In an inflammatory response, endothelial cells are activated to up‐regulate a number of genes, including E‐selectin (ELAM‐1), VCAM‐1, ICAM‐1, interleukin (IL)‐1, IL‐8 and plasminogen activator inhibitor‐1 (PAI‐1). Very little is known about factors that regulate the activation process. We describe here that a heat‐stable protein, normally present in the α‐globulin fraction of serum, inhibits induced expression of E‐selectin, ICAM‐1, and VCAM‐1 in vitro and also impedes the accumulation of mRNA for these molecules. Inhibition of E‐selectin, the only gene tested in this respect, is at the level of transcription. At the same time, the α‐globulins do not, under the same conditions, repress mRNA accumulation for IL‐1, IL‐8, or PAI‐1. The effect of the inhibitor does not relate to constraints on function of nuclear‐factor χB, the induced activity of which is not interfered with at the early time points at which the suppression of these three genes is seen.
Exploring myelin basic protein for HLA class I‐binding sequences
Authors: Tanigaki Nobuyuki , Fruci Doriana , Groome Niegel , Butler Richard H. , Londei Marco , Tosi Roberto
Published: March 2007

In view of the increasing evidence of the involvement of CD8+ T cells in the pathogenesis of multiple sclerosis (MS), we have scanned the sequence of the myelin basic protein (MBP), using 162 overlapping nonapeptides, for HLA‐class I binding sites. Peptide binding was measured using the recently reported HLA class I α‐chain‐refolding assay, and the following HLA allelic products were analyzed: HLA‐A2 (*0201, *0204), B27 (*2705), B35, B51, and B62. A considerable number of binding peptides were distinguished for each of the allelic products tested. In addition, three interesting points emerged. The first was the identification of several binding peptides which did not contain the known anchor motifs. The second was the evidence that several peptides showed a promiscuous binding profile, being able to bind to different HLA class I molecules that were either allelic or non allelic. The third was that in several cases two consecutive peptides could bind to the same HLA molecule.
Homotypic aggregation of CD103 (αEβ7)+ lymphocytes by an anti‐CD103 antibody, HML‐4
Authors: Benmerah Alexandre , Badrichani Anne , Ngohou Kalyane , Mégarbané Bruno , Bègue Bernadette , Cerf‐Bensussan Nadine
Published: March 2007

One monoclonal antibody, HML‐4, directed against the αEβ7 integrin (CD103), an integrin preferentially expressed on human intestinal intraepithelial lymphocytes (IEL), induced the homotypic aggregation of IEL and of a CD103+ MOLT16 cell line. Aggregation was an active adhesion event dependent on an intact cytoskeleton, on tyrosine phosphorylation but not on activation of protein kinase C. It was blocked by four other anti‐CD103 antibodies but by none of the antibodies blocking known adhesion lymphocyte pathways. It was associated with a redistribution of the CD103 integrin in the areas of cell‐cell contacts. These results indicated that HML‐4zx‐induced homotypic adhesion was mediated via CD103 and resulted from the binding of the integrin to an as yet undefined ligand expressed by CD103+ cells. This ligand was distinct from the epithelial ligand of CD103: in contrast with homotypic adhesion, heterotypic adhesion of CD103+ MOLT16 cells on two epithelial intestinal cell lines (DLD1 and HT29) was dependent on the presence of divalent cations, was not enhanced by HML‐4, was inhibited by HML‐1 but not by the three other antibodies with an inhibitory effect on homotypic adhesion. Finally, the study of conjugates between CD103+ and CD103‐ sublines derived from the MOLT16 cell line suggested that HML‐4‐induced homotypic aggregation resulted from homophilic CD103‐CD103 interactions.
Vasoactive intestinal peptide specifically induces human IgA1 and IgA2 production
Authors: Kimata Hajime , Fujimoto Masayuki
Published: March 2007

The effects of vasoactive intestinal peptide (VIP) on human IgA1 and IgA2 production were studied. In unfractionated small resting B cells stimulated with anti‐CD40 monoclonal antibody (mAb), VIP induced IgA1 and IgA2 production without affecting the production of IgG1, IgG2, IgG3, IgG4, IgM, or IgE. When small B cells were separated into sIgA1+, sIgA2+, sIgA1‐ and sIgA2‐ B cells, anti‐CD40 mAb plus VIP induced IgA1 and IgA2 production by surface IgA1‐ (sIgA1‐) and sIgA2‐ B cells, respectively, while having no effect on sIgA1+ and sIgA2+ B cells. This induction by VIP was specific, since anti‐CD40 mAb plus other neuropeptides, i.e., somatostatin or substance P, had no effect, and moreover, the induction was specifically blocked by a VIP antagonist. Further, anti‐CD40 mAb plus various cytokines, including interleukin (IL)‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐10, transforming growth factor‐β, low molecular weight B cell growth factor, and interferon‐γ, did not induce IgA1 and IgA2 production by sIgA1‐ and sIgA2‐ B cells, respectively. These results indicate that in the presence of anti‐CD40 mAb, VIP induces IgA1 and IgA2 production by isotype switching.
T cell clones expressing the natural killer cell‐related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function
Authors: Ferrini Silvano , Cambiaggi Anna , Meazza Raffaella , Sforzini Sabrina , Marciano Sabrina , Mingari Maria Cristina , Moretta Lorenzo
Published: March 2007

A new anti‐p58 monoclonal antibody (mAb), termed CH‐L, has been used to characterize a minor subset of T lymphocytes co‐expressing p58 and CD3 molecules. In two‐color immunofluorescence analysis, CH‐L+CD3+ cells represented 0.5 to 6% of the peripheral blood lymphocytes (in 20 healthy donors). Clonal analysis showed that most CD3+CH‐L+ T cell clones expressed the CD8+4− T cell receptor (TcR) α/β+ phenotype, while only a few were CD8−4+ TcR α/β+, CD8−4− TcR α/β+ or CD8−4− TcR γ/δ+. Western blot analysis indicated that the CH‐L mAb identifies the same 56‐58‐kDa diffuse band in both T and natural killer cell (NK) clones. A minority of T cell clones also expressed other NK‐related markers such as CD16, CD56 and CD94 and two clones also reacted with the anti‐p58 mAb EB6. Interestingly, most clones displayed cytolytic activity in an anti‐CD3 mAb‐triggered redirected killing assay against the Fcδ receptor+ P815 target cells and NK‐like activity against K562 and Raji cells. In contrast, the IGROV‐1 ovarian carcinoma cell line was resistant to cytolysis by all of these clones. Since p58 molecules have previously been shown to exert regulatory functions on NK‐mediated lysis, we investigated whether anti‐p58 mAb could also influence cytotoxicity mediated by CD3+p58+ T lymphocytes. Lysis of P815 target cells, triggered by anti‐CD3 mAb, could be inhibited by anti‐p58 mAb in 8 out of 12 cytolytic clones tested, while 4 clones were not inhibited. In addition, anti‐p58 mAb enhanced the cytolytic activity of 3 clones against IGROV‐1 and of 4 other clones against Raji target cells. Taken together, these data indicate that p58+ T cells express heterogeneous phenotypes and different forms of TcR and, in most instances, display cytolytic functions. Perhaps more importantly, the p58 molecule appears to modulate the cytolytic activity triggered via the CD3/TcR complex.
Healthy monozygous twins do not recognize identical T cell epitopes on the myelin basic protein autoantigen
Authors: Shanmugam Ananth , Copie‐Bergman Christiane , Hashim George , Rebibo Danielle , Jais Jean Philippe , Bach Jean‐Francois , Bach Marie Anne , Tournier‐Lasserve Elisabeth
Published: March 2007

The T cell response against myelin basic protein (MBP) has been extensively studied in humans because of its putative role in the pathophysiology of multiple sclerosis (MS). Higher concordance rates in monozygous twins as well as an increased risk in relatives suggest the role of genetic factors in MS susceptibility. Very little is known about the shaping of T cell repertoire towards self antigens in humans and their contribution to disease susceptibility in autoimmune disorders. Here we report the comparative T cell epitope recognition patterns towards the MBP auto‐antigen in healthy identical twins. We have established MBP‐specific T cell lines from eight sets of twins and characterized their fine epitope specificity. Intra‐pair comparison showed the co‐existence of shared as well as distinct epitopes in six of eight pairs and a complete absence of concordant epitope recognition within two other pairs. These findings indicate that important differences in T cell repertoires against a self antigen may be observed between genetically identical healthy individuals, rendering difficult the interpretation of the differences which may be observed between identical twins discordant for an autoimmune disease.
Major histocompatibility complex regulation of T helper functions mapped to a peptide C terminus that controls ligand density
Authors: Murray Joseph S. , Ferrandis‐Edwards Denise , Wolfe Curtis J. , Schountz Tony
Published: March 2007

The functional status (Th1‐ versus Th2‐like) of CD4 T cells primed against human collagen type IV (hCol IV) or a single 30mer peptide from the α2 chain of this molecule is predicted by the major histocompatibility complex (MHC) class II (I‐A) genotype of the responding mice. H‐2s mice elicit Th1‐like cell‐mediated responses to these antigens, whereas Th2‐like humoral responses are primed in H‐2b,d,k mice. We now report that the ability of MHC to dictate T helper function in this system depends upon a single amino acid of the minimal α2(IV) peptide. The C terminus of this minimal (12mer) peptide is ‐G‐G‐P‐K, which is predicted to form a β‐turn. The present data demonstrate that the terminal lysine (K) stabilizes the immunogens full biological effects necessary for exclusive cellmediated responses in H‐2s mice. The lysine‐truncated (11mer) peptide with otherwise identical sequence effectively primes T helper function in both H‐2b and H‐2s genotypes. Most importantly, our direct analysis of these peptides' presentation by live antigen‐presenting cells (APC) reveals that the 12mer is bound at a log higher density on H‐2s APC than on H‐2b APC, and that the 11mer is presented at an equally low relative density on APC from both genotypes. In vitro analyses of 12mer/11mer cross‐reactive Th clones demonstrate that I‐As restricted clones require about 1–2 log lower doses of 12mer peptide than 11mer peptide to stimulate equivalent thymidine incorporation and cytokine release. By contrast, I‐Ab‐restricted (12mer/11mer cross‐reactive) Th clones show no preference for the 12mer and require relatively high peptide doses similar to those required to stimulate the I‐As clones with the 11mer peptide. Thus, the peptide dose requirements of Th clones reflect the high density of presentation associated with the 12mer: I‐As ligand. Taken together, the results directly support the role of ligand density as an important control point in the functional decision of CD4 T cells.
Interleukin‐1 receptor expression by ovine afferent lymph dendritic cells: response to secondary antigen challenge
Authors: Fiskerstrand Carolyn E. , Hopkins John , Sargan David R.
Published: March 2007

Interleukin(IL)‐1 is thought to enhance the function of antigen presenting cells of the dendritic cell lineage. To investigate the interaction of IL‐1 and dendritic cells, recombinant ovine IL‐1α and IL‐1β have been used to determine IL‐1 receptor (R) expression on fresh dendritic cells (ALDC) collected from cannulated sheep pseudoafferent lymph ducts, both prior to and in response to localized ovalbumin challenge. Resting ovine ALDC express ∼ 510 IL‐1R per cell for IL‐1α (Kd ∼ 30 pM) and ∼ 350 IL‐1R/cell for IL‐β (Kd ∼ 160 pM). Saturation binding and in situ analyses show an initial transient but dramatic increase in IL‐1α binding to ALDC by 4 h in response to ovalbumin challenge of primed sheep. Maximal IL‐1R expression, reaching ⩾ 21700 IL‐1R/cell for IL‐1α detected by around 48 h, was followed by a gradual return to resting level by 8 days post challenge. Fewer than 0.5% of resting ALDC expressed IL‐1R but at least 5% of ALDC bound IL‐1α after ovalbumin challenge. There was no evidence of specific up‐regulation of receptors for IL‐1β on these cells. Fresh ovine alveolar macrophages, used as a positive control for specific IL‐1R expression, were found to express ∼ 2600 sites/cell for IL‐1α (Kd ∼ 56 pM) and 16500 sites/cell for IL‐1β (Kd ∼ 4.6 pM). In view of the differing IL‐1 binding characteristics displayed by the receptors on the two cell types, it is postulated that afferent lymph dendritic cells and macrophages are not expressing the same form of IL‐1R.
Development of antibody diversity in single germinal centers: selective expansion of high‐affinity variants
Authors: Ziegner Mike , Steinhauser Gudrun , Berek Claudia
Published: March 2007

In a T cell‐dependent immune response the microenvironment of the germinal center plays a crucial role in the affinity maturation of the antigen‐specific immunoglobulins. In order to look at the development of antibody diversity we have isolated single germinal centers and sequenced light chains characteristic of 2‐phenyl‐oxazolone (phOx)‐specific antibodies. Fourteen days after immunization we can demonstrate various stages of intraclonal diversity. There are germinal centers where B cells are practically unmutated, suggesting that in these cases a substantial clonal expansion has taken place prior to the activation of the hypermutation mechanism. In other germinal centers, sequences with a low number of randomly distributed somatic mutations were observed, indicating that these changes have been introduced recently and/or that they fail to generate high‐affinity variants and hence provide no basis for affinity selection. Finally, germinal centers are found in which practically all sequences carry the amino acid substitutions characteristic of the high affinity phOx antibodies. In these latter cases the high‐affinity variants have been preferentially expanded. We conclude that affinity selection is a process that operates right from the beginning of germinal center development. Those B cells with a relative high affinity for the antigen gain a proliferative advantage over other cells and will dominate the response and these are the cells which will be selected to differentiate into memory cells.
Presentation of exogenous antigens by macrophages: analysis of major histocompatibility complex class I and II presentation and regulation by cytokines
Authors: Magdalena  , Kovacsovics‐Bankowski  , Rock Kenneth L.
Published: March 2007

There is an antigen presenting cell (APC) in the lymphoid organs capable of presenting exogenous antigen (Ag) with major histocompatibility complex (MHC) class I molecules. This study was initiated to isolate clones of these APC to definitively establish their phenotype and to further study their properties. Murine bone marrow macrophages (BM MΦ) were immortalized by overexpressing myc and raf oncogenes. Five BM MΦ cell lines were generated that are phagocytic and expressed at their surface MΦ differentiation Ag. All five cell lines processed and presented exogenous ovalbumin (OVA) with MHC class I molecules. They all presented OVA‐linked to a phagocytic substrate 102–104‐fold more efficiently than soluble Ag. Clonal isolates of two of the MΦ cell lines had an identical phenotype and functional properties as the uncloned lines. These results definitively establish that MΦ are APC with the capacity of presenting exogenous Ag with MHC class I molecules. Interferon (IFN)‐γ interleukin‐4, granulocyte‐macrophage colony stimulating factor and lipopolysaccharide either alone or in combination induced little or no augmentation and in some cases decreased presentation of exogenous OVA with MHC class I. In contrast, all of MΦ activating factors increased MHC class I expression. Moreover, IFN‐γ increased the presentation of cytosolic OVA, demonstrating differences between the presentation of cytosolic Ag versus exogenous Ag with MHC class I. Finally, some lines constitutively processed and presented exogenous OVA with MHC class II while others only presented after stimulation with IFN‐γ. These results demonstrate that the pathways involved in the presentation of exogenous Ag with MHC class I and class II are independently regulated and that a cloned cell is capable of presenting exogenous Ag through both pathways.
Increased Fas antigen expression in murine retrovirus‐induced immunodeficiency syndrome, MAIDS
Authors: Hiromatsu Kenji , Aoki Yoshiyasu , Makino Masahiko , Matsumoto Yoshihiro , Mizuochi Toshiaki , Gotoh Yoshitaka , Nomoto Kikuo , Ogasawara Jun , Nagata Shigekazu , Yoshikai Yasunobu
Published: March 2007

The Fas antigen (Fas), which is a cell surface protein belonging to the tumor necrosis factor receptor family, mediates apoptosis. To assess the contribution of Fas to the pathogenesis of retrovirus‐induced immunodeficiency, we examined the kinetics of Fas expression on the lymphocytes during the course of murine acquired immunodeficiency syndrome (MAIDS) induced by a defective LP‐BM5 murine leukemia virus. The Fas‐positive cells were increased in proportion both in αβ T cells and B cells with the progression of MAIDS. The appearance of Fas‐positive cells in αβ T cells preceeded those in B cells during the course of MAIDS. Among αβ T cells, about half of the Thy1.2+ αβ T cells were positive for Fas, while almost all of Thy1.2− CD4+ αβ T cells were of the Fas‐positive phenotype. The Fas‐positive cells in MAIDS mice, especially unique Thy1.2−CD4+ αβ T cells, were easily rendered apoptotic by stimulation via Fas, indicating that Fas expressed on the lymphocytes is functional. Furthermore, concomitant infection with Mycobacterium avium in MAIDS mice caused a marked increase in Fas‐positive cells accompanied by a severely impaired T cell reactivity to polyclonal stimuli. Taken together, these results suggest the possible participation of the Fas system in the pathogenesis of retrovirus‐induced immunodeficiency.
Adhesion molecules from the LFA‐1/ICAM‐1, 3 and VLA‐4/VCAM‐1 pathways on T lymphocytes and vascular endothelium in Graves' and Hashimoto's thyroid glands
Authors: Marazuela Mónica , Postigo Antonio A. , Acevedo Agustín , Díaz‐González Federico , Sánchez‐Madrid Francisco , De Landázuri Manuel O.
Published: March 2007

Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and, subsequently, their interaction with thyrocytes and extracellular matrix proteins. A number of different ligand molecules have been identified to mediate the interaction between EC and leukocyte subpopulations. In this study, we examined by flow cytometry and immunohistochemical techniques, the expression of integrin receptors and their counter‐receptors by infiltrating lymphocytes and vascular endothelium in thyroid glands from patients with Graves' disease (GD) and Hashimoto's thyroiditis (HT). A high proportion of GD intrathyroidal T lymphocytes expressed the CD69 and gp95/85 (Ea2) activation antigens as well as an increased number of LFA‐αL, VLA‐α1, ‐α4, ‐α5, and ‐β1 integrin receptors, as compared with peripheral blood T lymphocytes from the same patients. The expression of intercellular adhesion molecule (ICAM)‐1 was increased in EC from GD and HT thyroids. In addition, an up‐regulated de novo expression of vascular cell adhesion molecule (VCAM)‐1 was found in EC in GD and HT thyroids, with no reactivity in control thyroids. Dendritic cells in thyroid lymphoid follicles were also positive for ICAM‐1 and VCAM‐1. In addition, most of intrathyroidal mononuclear cells expressed the ICAM‐3 adhesion molecule. This enhanced expression of ICAM‐1 and VCAM‐1 by thyroid EC in GD and HT may reflect their ability to regulate leukocyte trafficking and activation by means of the expression of specific ligand molecules. Our data suggest that both the LFA‐1/ICAM‐1, ICAM‐3 and VLA‐4/VCAM‐1 pathways could play a relevant role in localizing and perpetuating the autoimmune response in the thyroid gland in autoimmune thyroid disorders.
Human IgG isotype‐specific amino acid residues affecting complement‐mediated cell lysis and phagocytosis
Authors: Brekke Ole Henrik , Michaelsen Terje E. , Aase Audun , Sandin Randi H. , Sandlie Inger
Published: March 2007

In this report we describe the construction of anti‐5‐iodo‐4‐hydroxy‐3‐nitrophen‐acetyl (NIP) mouse/human immunoglobulin (Ig)G4 chimeric molecules with altered amino acid residues in the CH2 domain. Three mutants are described: Gln‐268 is substituted by His in γ4 Q268H, Ser‐331 is substituted by Pro in γ4 S331P, and in γ4 Q268H/S331P both residues are substituted. The ability of the mutant molecules to induce complement‐mediated cell lysis (CML) and phagocytosis by FcγRII‐ and FcγRIII‐bearing polymorphonuclear leukocytes (PMN) were measured. In CML, γ4 Q268H was inactive, but both γ4 S331P and γ4 Q268H/S331P were active provided that the antigenic density on the target cells was high. In phagocytosis mediated by PMN, the mutants γ4 S331P and γ4 Q268H/S331P were both active only when complement was introduced. γ4 Q268H was not active in phagocytosis under any conditions. We conclude that His‐268 in human IgG molecules does not modulate CML activity or phagocytosis mediated by FcγRII and/or FcγRIII. Pro‐331 rescues CML activity in IgG4 molecules when the epitope density on the target cells is high, but does not affect FcγRII/FcγRIII‐mediated phagocytosis. In this manner the mutants γ4 S331P and γ4 Q268H/S331P mimic human IgG2. This could indicate a structural similarity between IgG2 and these mutant molecules that distinguish them from both IgG1 and IgG3.
Membrane CD45R isoform exchange on CD4 T cells is rapid, frequent and dynamic in vivo
Authors: Sparshott Sheila M. , Bell Eric B.
Published: March 2007

CD4 T cells bearing high (240–190 kDa) and low (180 kDa) molecular mass isoforms of the leukocyte common antigen CD45 define functionally distinct subsets which have been equated with naive and memory T cells. In the rat, CD4 T cells expressing a high molecular mass isoform [identified by monoclonal antibody MRC‐OX22 (anti‐CD45RC)] exchange this for the 180 kDa molecule (CD45RC−) when stimulated by antigen. Here we show, by transferring mature allotype‐marked CD45RC− CD4 T cells (depleted of immature Thy‐1+ CD45RC− recent thymic emigrants) into normal euthymic recipients, that many T cells re‐express the high molecular mass isoform in less than 6 h. By 24 h, 30–60% of CD45RC− CD4 T cells became CD45RC+; within a week the entire cohort appeared to exchange the low for the high molecular mass isoform. Isoform exchange was dynamic and many CD4 T cells returned once again to the CD45RC− state. CD45RC− CD4 T cells declined in number more rapidly than the CD45RC+ subset after transfer. The results suggest that CD45R isoforms distinguish between resting T cells (CD45RC+) and those which have encountered antigen in the recent past. CD45R isoforms would appear to be unsuitable markers of naive and memory T cells.