bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of the site of the IgG molecule that regulates maternofetal transmission in mice
Authors: Medesan Corneliu , Radu Caius , Kim Jin‐Kyoo , 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 mouse IgG1 (mIgG1) molecule that is involved in the intestinal transfer of recombinant Fc hinge fragments in neonatal mice. This site encompasses Ile‐253, His‐310, Gln‐311, His‐433 and Asn‐434, localized at the CH2‐CH3 domain interface and overlapping with the staphylococcal protein A‐binding and catabolic sites. In the present study, the effect of these mutations on the maternofetal transfer of Fc hinge fragments has been studied. Experiments to analyze transfer of radiolabeled Fc hinge fragments from the circulation of 15–18 day pregnant mice to fetuses in utero demonstrate that the mutations affect the maternofetal transmission in a way that correlates closely with the effects of the mutations on intestinal transfer and catabolism. The studies indicate that the neonatal Fc receptor, FcRn, is involved in transcytosis across both yolk sac and neonatal intestine in addition to the regulation of IgG catabolism.
Adult bone marrow contains precursors for CD5+ B cells
Authors: Huang Christene A. , Henry Cassis , Iacomini John , Imanishi‐Kari Thereza , Wortis Henry H.
Published: March 2007

To determine directly whether B cell precursors of adult origin are capable of generating CD5+ B cells, we reconstituted neonatal C3H.SCID mice with adult C57BL/6 bone marrow and analyzed splenic B cells 10 months later. Surface staining and flow cytometry revealed that the B cells were of donor origin and that 30% were CD5+. This confirms that in vivo generated CD5+ B cells can be adult derived. After anti‐IgM (but not lipopolysaccharide) stimulation in vitro, virtually all of the B cells from the bone marrow‐reconstituted mice expressed surface CD5. Sequence analysis of expressed VHDJH genes from the CD5+ B cells present after anti‐IgM stimulation revealed a high frequency of N nucleotide addition in CDR3 regions. The presence of N nucleotides indicates that these sequences were derived from CD5+ B cells of adult origin rather than from long‐lived fetal precursor B cells present in either the adult bone marrow at the time of transfer or adult spleen. These experiments demonstrate conclusively that adult bone marrow contains precursors for CD5+ B cells and that unlike fetal liver‐derived precursors these express terminal deoxynucleotidyl transferase.
Diversion of CD4+ T cell development from regulatory T helper to effector T helper cells alters the contact hypersensitivity response
Authors: Diiulio Nancy A. , Xu Hui , Fairchild Robert L.
Published: March 2007

Cutaneous sensitization to reactive haptens and subsequent challenge results in a T cell‐mediated response, contact hypersensitivity (CHS). Recent results from this laboratory have indicated that hapten sensitization induces two populations of reactive T cells: CD8+ T cells producing interferon (IFN)‐γ which mediate the response and CD4+ T cells producing interleukin (IL)‐4 and IL‐10 which negatively regulate the magnitude and duration of the response. Since CD4+ T cell development to either IFN‐γ‐ (Th1) or IL‐4/IL‐10‐ (Th2)‐producing cells is dependent upon the cytokine environment during antigen priming, we hypothesized that CD4+ T cell induction in a Th1‐promoting environment would not only alter the CD4+ T cell cytokine‐producing phenotype but also the course of the CHS response. Administration of the Th1‐promoting cytokine IL‐12 during hapten sensitization resulted in a CHS response of greater magnitude following challenge and extended the duration of the response. In hapten‐sensitized mice depleted of CD8+ T cells, treatment with IL‐12 induced effector CD4+ T cells. Histological examination of challenged ear tissue from these mice indicated minimal edema and an acute mononuclear cell infiltration more typical of classical delayed‐type hypersensitivity than CHS. Hapten‐primed CD4+ T cells from IL‐12 treated, sensitized mice produced IFN‐γ, but not IL‐4 in response to T cell receptor‐mediated stimulation. Use of neutralizing anti‐IFN‐γ antibody indicated that IL‐12 not only directly promoted Th1 development but also indirectly inhibited Th2 development through stimulation of IFN‐γ production at the time of hapten sensitization. Overall, these results demonstrate that diversion of CD4+ T cell development to Th1 effector cells rather than to Th2 cells alters the efferent nature of CHS and removes a primary regulatory mechanism of the immune response.
Plasmid DNA and protein vaccination of mice to the outer surface protein A of Borrelia burgdorferi leads to induction of T helper cells with specificity for a major epitope and augmentation of protective IgG antibodies in vivo
Authors: Zhong Weimin , Wiesmüller Karl‐Heinz , Kramer Michael D. , Wallich Reinhard , Simon Markus M.
Published: March 2007

Plasmid DNA‐based vaccination is an efficient way to evoke various forms of protective immunity in laboratory animals. Our previous experiments have shown that mice immunized with either plasmid DNA encoding the outer surface lipoprotein A (pOspA) of Borrelia burgdorferi or the respective lipoprotein (Lip‐OspA) produce protective antibodies against subsequent challenge with virulent spirochetes. In the present study, we compared the specificity and function of T cells generated in AKR/N mice previously immunized to either pOspA or Lip‐OspA. T cell populations derived by either of the two protocols consistently responded by proliferation in vitro to one (residues 186–203; B4) out of a panel of 27 overlapping 20‐mer peptides spanning the entire OspA molecule of strain ZS7. B4 was shown to express allele‐specific ligand motifs for I‐Ek. Most of the other peptides produced variable and much less pronounced or marginal proliferative T cell responses. T cells reactive to B4 as well as to some minor epitopes were CD4+CD8− T cells which produced IFN‐γ but no detectable IL‐4 upon antigen stimulation in vitro Priming of AKR/N mice with B4 but not with inactive peptides of OspA led to an enhanced production of IgG antibodies, mainly of the IgG1 isotype, including those to a prominent protective epitope (LA‐2) upon subsequent challenge with Lip‐OspA or intact spirochetes. The data demonstrate that both plasmid DNA and protein immunization with OspA results in T cell responses with specificity for a dominant OspA epitope and suggest that priming of mice with immunodominant peptides accelerates the appearance of protective antibodies in vivo. The identification of T helper cell epitopes relevant for the induction of protective antibodies will also facilitate the design of more potent vaccines against Lyme disease.
IsXenopusIgX an analog of IgA?
Authors: Mußmann Rainer , Du Pasquier Louis , Hsu Ellen
Published: March 2007

Using class specific monoclonal antibodies we analyzed the tissue distribution of B cells expressing the three immunoglobulin (Ig) isotypes (IgM, IgX, IgY) inXenopus. Large numbers of IgM‐ and IgX‐, but not IgY‐, positive B cells are located in the gut epithelium of the intestine. In this organ up to 60% of all B cells can be IgX positive, while in the spleen or liver they are hardly detectable. The majority of IgX‐producing cells resemble plasma cells. IgY‐producing cells are found in the liver and spleen but not in the intestine. In contrast to IgY, the expression of IgM and IgX is thymus independent. Upon systemic immunization, a several‐fold increase of specific IgM and IgY, but not IgX, antibodies was detected in the sera. This and its association with the mucosae of the intestine resembles results reported for mammalian IgA; therefore, IgX ofXenopusmight be considered an analog of IgA.
Interleukin‐10 and interleukin‐4 have similar effects on hapten‐specific primary antibody responses to penicillin in vivo
Authors: Kerdine Saadia , Pallardy Marc , Lamanètre Sandrine , Bertoglio Jacques , Lebrec Hervé
Published: March 2007

Interleukin (IL)‐10 was initially recognized on the basis of its capacity to inhibit production of interferon (IFN)‐γ by T helper (Th)1 lymphocytes; we examined whether this cytokine can bias the primary antibody (Ab) response to the hapten penicillin. We previously reported that BALB/c and SJL mice develop different responses to benzylpenicillin coupled to tetanus toxoid (BPO‐TT). The response of SJL mice was characterized as Th2 on the basis of early and high IL‐4 mRNA expression and production of BPO‐specific Ab of the IgG1 isotype. In contrast, the response of BALB/c mice was characterized as Th1 on the basis of delayed and weaker IL‐4 mRNA expression associated with high anti‐BPO IgG2a production (Kerdine, S. et al., Mol. Immunol. 1996. 33: 71). In this report, we demonstrate that in naive animals, the level of expression of IL‐10 mRNA in LN cells was high in SJL and barely detectable in BALB/c. In addition, injection of BPO‐TT resulted in rapid and large increase of IL‐10 mRNA expression in SJL. Neutralization of IL‐10 in vivo promoted the production of BPO‐specific IgG2a in SJL, and injection of IL‐10‐CHO cells inhibited BPO‐specific IgG2a production in BALB/c. Neutralization on administration of IL‐10 had effects very similar to neutralization or administration of IL‐4. However, co‐neutralization of IL‐10 and IL‐4 in SJL did not evidence additive or synergistic effects of the two cytokines. Administration of IL‐10 or IL‐4 in BALB/c inhibited BPO‐TT‐induced expression of IL‐12 p40 mRNA without modulating IFN‐γ mRNA. Together, these data demonstrate that endogenous production of IL‐10 regulates the production of IgG2a Ab in response to BPO‐TT and that IL‐10, like IL‐4, is critical for controlling primary responses to antibiotics which behave as haptenic compounds.
Homeostatic regulation of CD8+ T cells after antigen challenge in the absence of Fas (CD95)
Authors: Zimmermann Christine , Rawiel Marlies , Blaser Claudine , Kaufmann Martina , Pircher Hanspeter
Published: March 2007

The role of Fas in the homeostatic regulation of CD8+ T cells after antigen challenge was analyzed in the murine model of lymphocytic choriomeningitis virus (LCMV) infection. Mice homozygous for the lpr mutation and carrying T cell receptor (TCR) αβ transgenes specific for the LCMV glycoprotein peptide aa 33–41 in the context of H‐2Db were used. Five main results emerged: first, development of lymphadenopathy and of CD4−CD8− double‐negative B220+ T cells in lpr mice was not inhibited by the αβ TCR transgenes; second, tolerance induction and peripheral deletion of CD8+ T cells induced by LCMV glycoprotein peptide injection was independent of Fas expression; third, clonal down‐regulation of Fas‐deficient TCR‐transgenic CD8+ T cells after acute LCM virus infection was identical to the decline of transgenic T cells expressing Fas; fourth, in vivo activated CD8+ effector T cells from TCR transgenic and TCR‐lpr/lpr mice were equally susceptible to activation‐induced cell death in vitro; and fifth, transgenic effector T cells from lpr/lpr mice were cleared in the declining phase of the immune response in vivo without giving rise to CD4−CD8− double‐negative T cells. Taken together, these data suggest that the homeostatic regulation of CD8+ T cells after antigen challenge in vivo is regulated by mechanisms that do not require Fas.
CD40 signaling induces interleukin‐4‐independent IgE switching in vivo
Authors: Ferlin Walter G. , Severinson Eva , Ström Lena , Heath Andrew W. , Coffman Robert L. , Ferrick David A. , Howard Maureen C.
Published: March 2007

T cell‐deficient T cell receptor (TCR) β−/− x TCR δ−/− knockout mice lack circulating IgE and fail to produce antigen‐specific IgE in response to stimulation with T cell‐dependent antigens. We show here that these animals are able to produce significant levels of circulating polyclonal IgE when injected with an agonistic anti‐mouse CD40 monoclonal antibody. CD40‐mediated induction of circulating polyclonal IgE in T cell‐deficient mice was only partially reduced when the animals were co‐treated with neutralizing anti‐interleukin‐4 (IL‐4) antibody. The IL‐4 independence of this response was further supported by experiments showing that anti‐CD40 antibodies induced circulating IgE when injected into IL‐4 knockout mice, and sterile RNA ϵ transcript production when cultured with purified B cells from the same mice. These data strongly suggest that CD40 signaling causes IL‐4‐independent IgE switching in mice.
Monocytes induce E‐selectin gene expression in endothelial cells: role of CD11/CD18 and extracellular matrix proteins
Authors: Noble Karen E. , Panayiotidis Panayiotis , Collins Peter W. , Hoffbrand A. Victor , Yong Kwee L.
Published: March 2007

E‐selectin is an endothelium‐specific inducible adhesion molecule which binds several inflammatory cell types, including neutrophils, monocytes, natural killer cells and a subset of memory T cells. E‐selectin is important in the initial rolling interaction of these cells on inflamed endothelium. The transient kinetics of E‐selectin induction in vitro contrast with in vivo observations of prolonged expression of this adhesion molecule in chronic inflammation. This raises the possibility that signals generated within inflammatory tissues are more complex than the agonists used to activate endothelial cells in vitro. We investigated whether adhesive interactions with extravasating monocytes are able to provide activating signals that can induce E‐selectin expression on endothelium, and prolong the response to cytokine stimulation. We report that co‐culture with monocytes led to transcriptional activation of the E‐selectin gene in endothelial cells, and marked enhancement of the response to substimulatory concentrations of interleukin‐1. In addition, the presence of monocytes resulted in prolonged up‐regulation of E‐selectin. Induction of E‐selectin by monocytes was inhibited when cell contact between monocytes and endothelium was prevented (80 ± 8% inhibition, p < 0.001, n = 4). Monoclonal antibody (mAb) against tumor necrosis factor (TNF) was able to abolish 57.2 ± 9.7% of the response (p < 0.01, n = 4). The ability of adherent monocytes to induce sustained E‐selectin expression in endothelial cells could not be reproduced either by supernatants harvested from monocytes cultured for 18 h, or by maximal concentrations of TNF. The induction of E‐selectin in monocyte/endothelium co‐cultures was inhibited by mAb to CD11b, but not by those directed against VLA‐4 or L‐selectin. Extracellular matrix molecules may also play a role in adhesion‐dependent cellular activation, as inclusion of soluble collagen type I led to significant reduction in E‐selectin expression in monocyte/endothelium co‐cultures. We conclude that adhesive interactions between monocytes and endothelial cells provide a source of signals which influence the activation state of the endothelium, and consequently, the continued influx of inflammatory cells.
Phenotype and function of human B cells expressing CD70 (CD27 ligand)
Authors: Lens Susanne M. A. , De Jong Rolien , Hooibrink Berend , Koopman Gerrit , Pals Steven T. , van Oers Marinus H. J. , van Lier René A. W.
Published: March 2007

CD70, the cellular ligand of the tumor necrosis factor receptor family member CD27, can be found on a limited number of germinal center (GC) B cells in some tonsils, on scattered lymphocytes residing in secondary lymphoid organs, and on a fraction of the circulating B cell population. Due to the restricted expression of CD70 in vivo, we analyzed signals that determine CD70 expression levels and characterized the phenotype and function of CD70+ B cells. Expression of CD70 on B cells activated in vitro was found to be dependent on the continuous presence of a B cell antigen receptor cross‐linking agent, and induced or potentiated by CD40 ligation but was down‐modulated by the Th2 cytokines interleukin (IL)‐4 and IL‐13. Both in peripheral blood and tonsil cell suspensions, CD70+ B cell subpopulations were found to be enriched for CD27‐and IgG‐expressing cells, but contained less IgD+ B cells. Additional analysis of markers which define specific differentiation stages (Bm1‐5) of mature B cells within human tonsils did not place CD70‐expressing B cells in one of these subsets. Functional experiments revealed that whereas both CD70− and CD70+ B cells can secrete immunoglobulin after activation with a combination of Staphylococcus aureus Cowan strain I and IL‐2, only CD70+ B cells can produce large quantities of antibodies when stimulated in a T cell‐dependent fashion. Our combined data imply that CD70 is a marker for mature B cells which have recently been primed by antigen in vivo.
Potential allergens stimulate the release of mediators of the allergic response from cells of mast cell lineage in the absence of sensitization with antigen‐specific IgE
Authors: Machado Denise C. , Horton Donna , Harrop Richard , Peachell Peter T. , Helm Birgit A.
Published: March 2007

A number of structurally diverse antigens preferentially stimulate the synthesis of IgE antibodies, but no unifying principle has been proposed that explains the nature of isotype selection. In the present study, we show that common allergens present in bee venom, house dust mite emanations and parasite proteins induce mast cell and basophil degranulation and stimulate interleukin‐4 synthesis, and secretion in the absence of antigen‐specific IgE. These data point to a linkage between the initial activation of cells of the innate immune system and subsequent adaptive immune responses. They suggest that IgE‐independent mast cell and basophil degranulation is predictive of potential allergenicity and can be evaluated by means of a cellular assay. Our study indicates that non‐immunological degranulation by prototypic allergens, such as bee venom phos‐pholipase A2 or proteases associated with house dust mite emanations, is critically dependent on enzymatic activity. These findings have potentially important implications for vaccine design in allergic and parasitic disease.
Molecular cloning and chromosomal mapping of a novel human gene, ChemR1, expressed in T lymphocytes and polymorphonuclear cells and encoding a putative chemokine receptor
Authors: Samson Michel , Stordeur Patrick , Labbé Olivier , Soularue Pascal , Vassart Gilbert , Parmentier Marc
Published: March 2007

We describe the cloning of a human gene, named ChemR1, encoding a new putative chemokine receptor sharing 48% identity with CC‐chemokine receptor (CCR)4 and 44% identity with CCR1. It displays four extracellular cysteines that are conserved among all other chemokine receptors. ChemR1 transcripts were detected by Northern blotting in the T lymphoblastic cell lines Jurkat and MOLT‐4, but not in the pre‐B lymphoblastic cell line JM‐1. ChemR1 receptor transcripts were also detected by reverse transcription and polymerase chain reaction analysis in unstimulated CD4+ and CD8+ T cells and polymorphonuclear cells prepared from peripheral blood. The chromosomal localization was performed by radiation hybrid mapping and testing of a panel of yeast artificial chromosome clones. This allowed the assignment of the ChemR1 receptor gene to the p21.3‐24 region of human chromosome 3, in close proximity with the functionally characterized CCR. Future work is required to identify the ligand(s) of this new chemokine receptor and to define its role in the recruitment of white blood cell populations.
Spontaneously produced anti‐DNA/DNase I autoantibodies modulate nuclear apoptosis in living cells
Authors: Madaio Michael P. , Fabbi Marina , Tiso Micaela , Daga Antonio , Puccetti Antonio
Published: March 2007

Apoptosis has received increased attention over the past decade, and it is established as an essential process in physiological and disease states. Much effort has been devoted to understanding the intracellular mechanisms culminating in apoptosis; intense investigation has also focused on its role during inflammation. Despite these efforts, these events remain incompletely understood. It has been suggested that the Ca2+‐ and Mg2+‐dependent endonuclease that mediates DNA fragmentation is DNase I; however, the precise role of DNase I during apoptosis has been debated. Recent observations using anti‐DNA antibodies derived from autoimmune mice (MRL‐lpr/lpr) provided both the means and the reagents to approach these issues in a more direct manner. We previously discovered that many anti‐DNA antibodies cross‐react with DNase I, and a subset of these Ig inhibited DNase I enzymatic activity in vitro. Serendipitously, in separate studies, a subset of these antibodies were observed to enter and localize within the nuclei of living cells. The aim of the present investigation was to determine whether these nuclear‐localizing anti‐DNA antibodies could interact with DNase I in living cells. We found that, once internalized, these autoantibodies bound DNase I and inhibited activity of the enzyme. Furthermore, living cells containing the intracellular antibodies appeared resistant to apoptotic stimuli; both morphological features of nuclear apoptosis and DNA fragmentation were inhibited. These results support a pivotal role for DNase I in apoptosis, and they provide a novel paradigm for autoantibody‐mediated inflammatory disease.
Direct cell/cell contact with stimulated T lymphocytes induces the expression of cell adhesion molecules and cytokines by human brain microvascular endothelial cells
Authors: Lou Jinning , Dayer Jean‐Michel , Grau Georges E. , Burger Danielle
Published: March 2007

Upon inflammation, stimulated, but not resting T lymphocytes cross the blood‐brain barrier and migrate into the central nervous system. This study shows that direct contact between stimulated T lymphocytes and human brain microvascular endothelial cells (HB‐MVEC) induces phenotypic and functional changes on the latter cells. Plasma membranes isolated from stimulated T lymphocytes (S‐PM) up‐regulated the expression of intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and E‐selectin on isolated HB‐MVEC. In addition, HB‐MVEC activated by S‐PM secreted interleukin (IL)‐6 and IL‐8. The levels of ICAM‐1, E‐selectin, IL‐6, and IL‐8 expressed in S‐PM‐activated HB‐MVEC were similar to those observed with 1000 U/ml tumor necrosis factor (TNF). In contrast, VCAM‐1 expression was 15% of that induced by TNF. Inhibitors of TNF diminished (≤ 45 %), but did not abolish the expression of cell adhesion molecules and IL‐6 induced by S‐PM, IL‐8 production being insignificantly affected (≤ 10 %). This suggests that membrane‐associated TNF was partially involved in HB‐MVEC activation. The present study demonstrates that stimulated T lymphocytes are able to activate HB‐MVEC upon direct cell contact. This novel mechanism of inducing the expression of cell adhesion molecules may prompt the initial adhesion of stimulated T lymphocytes to brain endothelium.
Modulation of Apo‐1/Fas (CD95)‐induced programmed cell death in myeloma cells by interferon‐α2
Authors: Egle Alexander , Villunger Andreas , Kos Marion , Böck Günther , Gruber Johann , Auer Bernhard , Greil Richard
Published: March 2007

The Apo‐1/Fas (CD95) antigen is known to be involved in the process of T cell‐mediated target cell killing and has recently been shown to be expressed on myeloma cell lines and native malignant plasma cells. Several cytokines have been reported to interfere with spontaneous and even Apo‐1/Fas‐induced apoptosis, but no attempt has been made yet to investigate these interactions and the possible underlying mechanisms in myeloma cells. Since in myeloma patients Interferon (IFN)‐α2 displays a profound therapeutic effect in vivo, which is usually attributed to its growth inhibitory and/or immunomodulatory capacity, we set out to study the potential interference of IFN‐α2 with Apo‐1/Fas‐induced apoptosis. Contrary to expectations, IFN‐α2 reduced the degree of apoptosis caused by the treatment of five Apo‐1/Fas‐sensitive myeloma cell lines with a Fas monoclonal antibody (mAb). Simultaneous application of IFN‐α2 and Fas mAb was superior to the prolonged (i.e. > 8 h) preincubation with the cytokine as far as inhibition of Apo‐1/Fas‐induced apoptosis was concerned. This effect of IFN‐α2 was neither explained by a down‐regulation of the Apo‐1/Fas receptor nor caused by modulation of the expression levels of c‐myc, bcl‐2‐, bcl‐xL, bax‐ or p53 genes. IFN‐α2 did not alter the Apo‐1/Fas‐induced activity of Mitogen‐activated protein kinase (MAPK) 1 and did not inhibit the Apo‐1/Fas‐mediated proteolytic cleavage of ADP‐ribosyltransferase, a substrate of Interleukin‐β1 converting enzyme (ICE) and homologues. However, activation of protein kinase C (PKC) by phorbol 12‐myristate 13‐acetate (PMA) mimicked the effects of IFN‐α2. Furthermore, the bis‐indolylmaleimide GF 109203X, a specific inhibitor of PKC, inhibited the effect of PMA as well as that of IFN‐α2 on Apo‐1/Fas‐induced apoptosis. These results point to a PKC‐dependent mechanism of transient interaction between the intracellular signaling along the IFN‐α2 and the Apo‐1/Fas pathway (downstream of MAPK signaling as well as of ICE homologues), which becomes exhausted by prolonged stimulation with the cytokine. According to our data IFN‐α2, applied continuously and in high doses resembling the therapeutic situation in vivo, inhibits myeloma growth. However, based on the observed inhibitory effect of IFN‐α2 on Apo‐1/Fas‐induced apoptosis, a partial inhibition of the natural immune surveillance on myeloma cells by endog‐genous IFN‐α2 present in the bone marrow microenvironment of this malignancy should be investigated.
Mouse polymorphonuclear granulocyte binding to extracellular matrix molecules involves β1 integrins
Authors: Frieser Michael , Hallmann Rupert , Johansson Staffan , Vestweber Dietmar , Goodman Simon L. , Sorokin Lydia
Published: March 2007

The mechanism of adhesion of purified mouse polymorphonuclear granulocytes (PMN) to extracellular matrix proteins characteristic of basement membranes and the interstitium has been investigated and compared with the adhesion of a mouse progranulocytic cell line, 32DC13, and a mouse monocytic cell line, WEHI 78/24. All three cell types bound specifically to fibronectin and vitronectin to different degrees under different cellular activation states. 32DC13 bound to fibronectin and vitronectin strongly, and this binding increased upon cellular activation with phorbol 12‐myristate‐13‐acetate (PMA) but not with formyl‐Met‐Leu‐Phe. Only 32DC13 showed significant binding to laminin‐1. By contrast, WEHI 78/24 and PMN bound only fibronectin and vitronectin; this binding was weak and was altered only marginally upon activation with PMA. In the case of WEHI 78/24, a slight increase in adhesion both to fibronectin and to vitronectin was observed after cellular activation with PMA, while PMN adhesion to both substrates was slightly reduced. The mechanism of binding to fibronectin and vitronectin was similar in the three cell types. The integrin α5β1 mediated fibronectin adhesion, demonstrating for the first time the existence of a functionally active β1 integrin on mouse PMN. Vitronectin binding was mediated by αvβ3, as demonstrated by the ability of αv‐specific cyclic L‐Arg‐L‐Gly‐L‐Asp‐D‐Phe‐L‐Val (RGDfV) peptide (EMD66203), and anti‐β3 antibody to inhibit cell adhesion. 32DC13 adhesion to laminin‐1 was via the α6β1 integrin. None of the three cell types tested bound to the basement membrane proteins collagen type IV and perlecan, or to the interstitial stromal constituents tenascin, collagen types I, V and VI. Interestingly, perlecan and collagen type IV were found to repel all three cell types. The relative inability of PMN, WEHI 78/24, and 32DC13 to bind to extracellular matrix proteins characteristic of basement membranes and their ability to bind inflammatory markers of the interstitium is discussed with respect to leukocyte extravasation processes.
Induction, regulation, and function of soluble TRAP (CD40 ligand) during interaction of primary CD4+ CD45RA+ T cells with dendritic cells
Authors: Ludewig Burkhard , Henn Volker , Schröder Jens M. , Graf Daniel , Kroczek Richard A.
Published: March 2007

To assess the induction, regulation, and the relative roles of cell surface tumor necrosis factor‐related activation protein (TRAP; CD40 ligand) and the soluble form of TRAP (sTRAP) in the initial phase of T cell activation, primary CD4+ CD45RA+ (naive) T cells were co‐cultured with mature Langerhans' cells (mLC) in the presence of superantigen. In this cell system, TRAP was very efficiently induced in T cells at both the mRNA and protein levels. After appearing on the cell surface, TRAP was rapidly down‐regulated by a mechanism triggered through interaction of TRAP with CD40 on mLC. Co‐culture of T cells with mLC led to the release of sTRAP, an 18‐kDa protein capable of binding to CD40. Experimental data strongly suggest that sTRAP is not released by proteolytic cleavage of TRAP on the cell surface, but is generated in an intracellular compartment. Release of sTRAP and induction of TRAP cell surface expression were found to be regulated independently. In terms of function, sTRAP cannot compete with cell surface TRAP for ligation of CD40 on mLC, indicating that sTRAP release is not a mechanism for termination of the TRAP/CD40 interaction. However, sTRAP on its own rapidly down‐regulates CD40 expression on mLC and has long‐lasting anti‐apoptotic effects on dendritic cells. Thus, we infer from our results obtained in vitro that primary activation of CD4+ T cells by dendritic cells in the lymphoid tissues leads to release of sTRAP, which may act on CD40+ bystander cells in a cytokine‐like fashion.
Cell division in the compartment of naive and memory T lymphocytes
Authors: Bruno Ludovica , von Boehmer Harald , Kirberg Jörg
Published: March 2007

Expression of activation markers and proliferative status were measured in peripheral CD4+ and CD8+ T cells of various T cell receptor (TCR)‐transgenic mice either before or after intentional antigenic stimulation. In the absence of intentional immunization, CD4+ T cells persisted as resting or partially activated and cycling cells depending on the specificity of their TCR. Similar results were obtained following transfer into T cell‐deficient recipients, i.e. T cells that were not cycling in situ did not cycle after transfer, whereas cells that were proliferating in situ also cycled after transfer. Thus, the TCR of some cells in the absence of intentional antigenic stimulation may bind to some unidentified ligand that does not induce tolerance, but rather slow expansion. In a different sort of experiment, activated T cells that were derived from noncycling naive T cells by deliberate antigenic stimulation continued to cycle slowly even a long time after transfer into antigen‐free recipients that did not induce proliferation of the naive cells. Thus, lymphokines or ligands that do not induce activation of naive T cells may be responsible for the maintenance of memory cells. Our experiments show that the latter does not depend on a second TCR expressed by the memory cells, since memory T cells from RAG‐2−/−TCR‐transgenic mice persisted to a similar extent.
Differential expression of CLIP: MHC class II and conventional endogenous peptide: MHC class II complexes by thymic epithelial cells and peripheral antigen‐presenting cells
Authors: Farr Andrew , Deroos Paul C. , Eastman Susan , Rudensky Alexander Y.
Published: March 2007

Major histocompatibility complex (MHC) class II molecules expressed by thymic epithelial cells are involved in positive selection of CD4 T cells, whereas the high‐avidity interaction of T cell receptors with the endogenous peptide : MHC class II complexes expressed on bone marrow (BM)‐derived antigen‐presenting cells (APC) and, to a lesser extent, on thymic epithelial cells mediate negative selection. To understand better the generation of the CD4 T cell repertoire both in the thymus and in the periphery we analyzed relative levels of expression of specific endogenous peptide: MHC class II complexes in thymic epithelial cells (TEC) and peripheral APC. Expression of Eα52–68: I‐Ab and class II‐associated invariant chain peptide (CLIP): I‐Ab complexes in thymic epithelial cells and in the bone‐marrow derived splenic APC, i.e. B cells, was studied using YAe and 30‐2 monoclonal antibodies which are specific for the corresponding complexes. To distinguish between expression of both complexes in radioresistant thymic epithelial elements and radiation sensitive BM‐derived APC, radiation BM chimeras were constructed. Using immunohistochemical and immunochemical approaches we demonstrated that the level of expression of Eα52–68:I‐Ab complexes in thymic epithelial cells is approximately 5–10 % of that seen in splenic cells whereas total class II levels were comparable. In contrast, CLIP: I‐Ab complexes are expressed at substantially higher levels in TEC vs. splenic APC. This result demonstrates quantitative differences in expression of distinct peptide: MHC class II complexes in thymic epithelial cells and peripheral splenic APC.
The role of tyrosine phosphorylation and PTP‐1C in CTLA‐4 signal transduction
Authors: Chambers Cynthia A. , Allison James P.
Published: March 2007

Recently, there has been increasing interest in the inhibitory regulators of lymphocyte activation, and in particular, the role of CD28 homologue CTLA‐4 in the regulation of T cell responses. Interaction of CTLA‐4 with B7 ligands inhibits T cell responses, including T cell proliferation and interleukin‐2 (IL‐2) secretion. The mechanism(s) responsible for CTLA‐4 signal transduction are unknown, but it has been suggested that tyrosine phosphorylation is involved. Here we demonstrate that phorbol ester phorbol 12‐myrislate 13‐acetate (PMA), which increases tyrosine phosphorylation by stimulating protein kinase C and p21ras, can overcome the CTLA‐4‐mediated inhibition of T cell proliferation. This provides the first functional evidence that tyrosine phosphorylation is involved in CTLA‐4 signal transduction. Most interestingly, CTLA‐4‐mediated inhibition of IL‐2 secretion was not influenced by the presence of PMA. Further, we demonstrate that CTLA‐4 cross‐linking inhibits proliferation and IL‐2 secretion of T cells from motheaten mice. These mice lack PTP‐1C, a tyrosine phosphatase which mediates in a number of lymphocyte inhibitory responses, indicating that PTP‐1C is not essential for CTLA‐4 signaling. Collectively, these results demonstrate that regulation of tyrosine phosphorylation plays a pivotal role in CTLA‐4 function, and that the inhibition of the transition from G0/G1 to the S phase of the cell cycle and the inhibition of IL‐2 secretion require distinct signaling pathways. These experiments provide a useful model system which can be used to elucidate the signaling pathways involved in CTLA‐4 function and to understand how CTLA‐4, CD28 and T cell receptor‐mediated signals are integrated in T cell responses to antigen.
Human leukocyte antigen phenotype imposes complex constraints on the antigen‐specific cytotoxic T lymphocyte repertoire
Authors: Burrows Scott R. , Silins Sharon L. , Cross Simone M. , Au Peh Chen , Rischmueller Maureen , Burrows Jacqueline M. , Elliott Suzanne L. , McCluskey James
Published: March 2007

The memory response to the immunodominant Epstein‐Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, is exceptionally restricted, being dominated by cytotoxic T lymphocytes (CTL) with a single, public T cell receptor (TCR). CTL clones that express this receptor fortuitously cross‐react with the alloantigen HLA B44. However, of the two major subtypes of this HLA, B*4402 and B*4403, that differ by a single amino acid, only the former is recognized by these mature CTL clones. Individuals heterozygous for HLA B8 and B*4402 use alternative TCR for the EBV determinant since the dominant TCR is potentially self‐reactive. We now demonstrate that this clonotype is also essentially absent from the repertoire of CTL directed against the viral epitope in seven from seven unrelated individuals heterozygous for HLA B8 and B*4403. Thus immune tolerance of these CTL recognizing HLA B*4402 is associated with expression of either B*4402 or B*4403. This suggests that tolerance in the human T cell compartment requires a lower threshold of recognition than for effector function, thus providing a buffer zone minimizing the risk of autoimmunity. These data also illustrate the potential for non‐restricting HLA molecules to bias dramatically the T cell repertoire used for specific immune responses. Such influences may be the basis of the “protective” effects of certain HLA alleles in susceptibility to autoimmune disorders.
A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells
Authors: Luhtala Marko , Lassila Olli , Toivanen Paavo , Vainio Olli
Published: March 2007

In this study we show the inheritance of a CD4+CD8+ peripheral T cell population in the H.B15 chicken strain. A large proportion of αβ T cells in peripheral blood (20–40%), spleen (10–20%) and intestinal epithelium (5–10%) co‐express CD4 and CD8α, but not CD8β. CD4+ CD8αα cells are functionally normal T cells, since they proliferate in response to mitogens and signals delivered via the αβT cell receptor as well as via the CD28 co‐receptor. These cells induce in vivo a graft versus host‐reaction, providing further evidence for their function as CD4+ T cells. The CD4+CD8αα T cell population was found in 75% of the first progeny and in 100% of further progenies, demonstrating that co‐expression of CD4 and CD8 on peripheral T cells is an inherited phenomenon. In addition, cross‐breeding data suggest a dominant Mendelian form of inheritance. The hereditary expression of CD8α on peripheral CD4+ T cells in chicken provides a unique model in which to study the regulation of CD4 and CD8 expression.
Cytotoxic T lymphocytes express a β3 integrin which can induce the phosphorylation of focal adhesion kinase and the related PYK‐2
Authors: Ma Eileen A. , Lou Olivia , Berg Nancy N. , Ostergaard Hanne L.
Published: March 2007

Fibronectin has been shown to stimulate tyrosine phosphorylation of a number of proteins in the 115–125 kDa range and facilitate degranulation by alloantigen‐specific cytotoxic T lymphocyte (CTL) clones in response to substimulatory amounts of anti‐CD3 or anti‐T cell receptor (TCR). The current study was initiated to further characterize integrin expression and usage by these CTL clones. We demonstrate that vitronectin and fibrinogen, but not laminin or collagen, are also able to both facilitate degranulation in the presence of substimulatory anti‐CD3 and stimulate tyrosine phosphorylation of these 115–125‐kDa proteins, with a 115‐kDa protein being the most prominently phosphorylated. These results implicate the expression and usage of the vitronectin receptor, α β3 integrin, by these CTL clones. We demonstrate by both flow cytometry and immunoprecipitation that CTL clones do in fact express β3 integrin. Immobilized antibody to β3 stimulates the phosphorylation of the 115–125‐kDa proteins, suggesting that engagement of β3 transmits the same signal into these cells as fibronectin or vitronectin. The fibronectin and vitronectin‐induced phosphorylation as well as adhesion to either fibronectin or vitronectin can be significantly inhibited with antibodies to β3 integrins. Finally, we are able to immunoprecipitate 115‐kDa proteins with antiserum to focal adhesion kinase and a related kinase, called PYK‐2, that becomes phosphorylated in response to vitronectin or immobilized anti‐β3. Taken together, these results demonstrate that CTL express and use β3‐integrins as signaling molecules which can augment TCR‐mediated stimulation.
Absolute dependence of T cell receptorhi cell generation and relative dependence of T cell receptorint cell generation on the thymus
Authors: Tsukahara Akihiro , Moroda Tetsuya , Iiai Tsuneo , Suzuki Susumu , Tada Takashi , Hatakeyama Katsuyoshi , Abo Toru
Published: March 2007

Recent evidence indicates that conventional T cells are generated by the mainstream of T cell differentiation in the thymus and acquire a high density of T cell receptor expression (i.e. TCRhi). In contrast, primordial T cells (or NK1.1+ T cells) are generated by the extrathymic pathways or an alternative intrathymic pathway and express an intermediate density of TCR (i.e. TCRint). To obtain further evidence, it was examined how thymus grafting influenced the distribution of T cell populations in athymic nude mice. When BALB/c nu/nu mice were engrafted with thymocyte‐depleted BALB/c+/+ fetal thymi, two changes emerged after grafting: nude mice generated TCRhi cells de novo in the periphery as well as in the grafted thymi, and the absolute number of interleukin‐2 receptor β chain+ TCRint cells increased prominently in number in the periphery. Among thymic hormones tested, the administration of thymosin α induced a slight expansion of CD3int cells in nude mice. To examine a possible interaction of TCRint cells with TCRhi cells in the periphery, B6 nu/nu mice (Ly5.2+) were injected with TCRhi cells purified from the spleen of B6 Ly5.1 congenic mice. In this case, TCRint (Ly5.2+) cells expanded well in all tested organs of nude mice. These results suggest that the generation of TCRhi cells is absolutely dependent on the thymus and that TCRint cells expand under the influence of the thymus (humoral) and due to interaction with thymus‐derived conventional T cells.
NFE, a new transcriptional activator that facilitates p50 and c‐Rel‐dependent IgH 3′ enhancer activity
Authors: Linderson Ylva , Cross Daniel , Neurath Markus F. , Pettersson Sven
Published: March 2007

The induction of immunoglobulin heavy chain (IgH) 3′ enhancer activity has been coupled to ligand/receptor‐dependent activation of resting B cells. To search for transcriptional target sites that account for this induction, extracts from lipopolysaccharide (LPS)‐stimulated B cells and cell lines were used. Here we describe, by gel‐retardation analysis, the identification of an NF‐χB site and an adjacent nuclear factor ets‐like (NFE) site in the 3′ enhancer. The NFE motif binds four protein complexes in resting B cell extracts, of which two are down‐regulated upon LPS stimulation. Gel shift‐shift experiments of the NF‐χB complexes with specific antibodies identified p50 and c‐Rel proteins to be the predominant factors in primary LPS‐stimulated cell extracts. Site‐directed mutagenesis of these motifs demonstrates that they contribute to part of the enhancer activity in plasma cells. One copy of the NFχB/NFE motifs, linked to a hetero‐logous reporter construct, displays lymphoid‐restricted reporter gene activity in transient transfection assays. Mutation of either site abrogates all promoter activity. Complementation experiments demonstrate that although p50 and c‐Rel expression vectors reconstitute transcription of an intact NF‐χB/NFE reporter construct in a dose‐dependent manner, mutation of the NFE site or the NF‐χB site abrogates essentially all transcriptional activity in both plasma cells and in COS cells. Taken together, we provide evidence for the existence of an activator, NFE, which in combination with the p50 and c‐Rel proteins, are part of the transcription factor machinery that regulates 3′ enhancer activity, and thus the control of the IgH locus in late B lymphocyte development.
Structural identification of the hematopoietic progenitor antigen ER‐MP12 as the vascular endothelial adhesion molecule PECAM‐1 (CD31)
Authors: Ling Vincent , Luxenberg Deborah , Wang Jack , Nickbarg Elliot , Leenen Pieter J. M. , Neben Steven , Kobayashi Michiko
Published: March 2007

The monoclonal antibody ER‐MP12 was recently described to recognize an antigen present on cell subpopulations of adult mouse bone marrow, including pluri‐potent hematopoietic stem cells. In an effort to understand the function of ER‐MP12 antigen in hematopoiesis, we used biochemical and physical methods to determine its identity. ER‐MP12 antigen was isolated by immunoprecipitation from FDCP‐1 cell membrane proteins, yielding a glycosylated 113‐kDa band upon analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thirteen peptides derived from trypsinized ER‐MP12 antigen were analyzed by electrospray ionization mass spectrometry and compared to a protein sequence database. The search revealed the identity of the ER‐MP12 antigen as platelet endothelial cell adhesion molecule‐1, CD31 (PECAM‐1). This result was subsequently confirmed by Edman sequencing of a single ER‐MP12 peptide fragment followed by comparison with PECAM‐1 sequence. In addition, flow cytometric analysis of bone marrow and embryonic stem cells revealed highly similar profiles between ER‐MP12 and CD31 (MEC 13.3 antibody)‐stained cells. The presence of PECAM‐1 on primitive hematopoietic stem cells supports the theory for the interaction of hematopoietic progenitor and stem cells with bone marrow stroma and transendothelial migration.
Sensitivity of Epstein‐Barr virus‐induced B cell tumor to apoptosis mediated by anti‐CD95/Apo‐1/fas antibody
Authors: Durandy Anne , Le Deist Françoise , Emile Jean‐François , Debatin Klaus , Fischer Alain
Published: March 2007

Epstein‐Barr virus (EBV) can induce uncontrolled B lymphocyte proliferation leading to fatal lymphoma in immunocompromised patients. The sensitivity to apoptosis of B lymphoid cell lines (LCL) derived from EBV‐induced lymphopro‐liferative disorders was investigated. In vitro and in vivo, these B LCL strongly express CD95/Apo‐1/fas antigen and undergo apoptosis upon stimulation with anti‐Apo‐1 monoclonal antibody. When inoculated into severe‐combined immunodeficient (scid) mice, human B cells lines developed into rapidly growing tumors. Administration of an agonistic anti‐Apo‐1 antibody significantly delayed tumor progression. Relapses were frequent, but were not caused by selection of resistant B cells, since B cells from relapsing tumors underwent apoptosis on re‐exposure. Induction of apoptosis by an anti‐C95/Apo‐1/fas‐specific antibody could be applied for therapy of EBV‐induced B cell tumors and contribute to our understanding of the mechanisms of T cell‐mediated elimination of EBV lymphomas in immunodeficient patients.
Comparison of cytotoxic T lymphocyte responses induced by peptide or DNA immunization: Implications on immunogenicity and immunodominance
Authors: Vitiello Antonella , Sette Alessandro , Yuan Lunli , Farness Peggy , Southwood Scott , Sidney John , Chesnut Robert W. , Grey Howard M. , Livingston Brian
Published: March 2007

To study the mechanisms that influence the immunogenicity and immunodominance of potential cytotoxic T lymphocyte (CTL) epitopes, we conducted a systematic analysis of the CTL response raised in HLA‐A*0201/Kb (A2/Kb) transgenic mice against the viral antigen, hepatitis B virus polymerase (HBV pol). From a pool of 26 nonamer peptides containing the HLA‐A*0201‐binding motif, we selected A2‐binding peptides, immunized A2/Kb animals, and tested the CTL raised against the peptide for recognition of HBV pol transfectants. Of nine immunogenic CTL epitopes, only four were recognized on HBV pol transfectants, whereas the other five were cryptic. Characterization of the peptide‐specific CTL lines indicated that crypticity may result from either poor processing or low T cell receptor (TCR) avidity. To identify the immunodominant epitopes, we determined the CTL specificities induced in A2/Kb animals in response to priming with HBV pol cDNA. We obtained a response against three epitopes that were contained with the set of four epitopes recognized by peptide‐specific CTL on HBV pol transfectants. Comparative analysis of cDNA priming and peptide priming revealed, therefore, the presence of a subdominant epitope. We conclude that for the HBV pol antigen, the repertoire of CTL specificities is shaped by major histocompatibility complex class I peptide binding capacity, antigen processing, and TCR availability.