bluuebunny/crossref_metadata_2025 huggingface dataset:
The thymus leukemia antigen binds human and mouse CD8.
Authors: Teitell M , Mescher M F , Olson C A , Littman D R , Kronenberg M
Published: June 2004

The thymus leukemia antigen (TLA) is a class Ib, or 'nonclassical' class I molecule, one of several encoded within the Tla locus of the mouse major histocompatibility complex (MHC). It structurally resembles the H-2K, D, and L class I transplantation antigens, which present processed peptides to cytotoxic T lymphocytes (CTLs). Although their function(s) are unknown, there has been recent speculation concerning the possibility that class Ib molecules may present antigens to T cells that express gamma delta T cell antigen receptors (TCRs). In this report, using both a cell-cell adhesion assay and adhesion of T lymphocyte clones to purified plate-bound TLA, we provide evidence that TLA can bind to both human and mouse CD8. We also show that a chimeric class I molecule containing the peptide antigen binding site of Ld and the alpha 3 domain, transmembrane, and cytoplasmic segments of TLA, can support a CD8-dependent immune response by CTLs. These results demonstrate for the first time binding of a class Ib molecule to CD8 with a functional outcome, as is observed for the class I transplantation antigens. The capacity to interact with CD8 has been conserved despite the extensive sequence divergence of TLA in the peptide antigen binding site, suggesting this interaction is highly significant. TLA is expressed by epithelial cells in the mouse small intestine. As these epithelial cells are in close contact with intestinal intraepithelial lymphocytes that are nearly all CD8+, and many of which express the gamma delta TCR, the data are consistent with the hypothesis that TLA is involved in antigen presentation, perhaps to gamma delta-positive lymphocytes in this site.
Regulation of cytokine production in the human thymus: epidermal growth factor and transforming growth factor alpha regulate mRNA levels of interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6 in human thymic epithelial cells at a post-transcriptional level.
Authors: Le P T , Lazorick S , Whichard L P , Haynes B F , Singer K H
Published: June 2004

Human thymic epithelial (TE) cells produce interleukin 1 alpha (IL-1 alpha), IL-1 beta, and IL-6, cytokines that are important for thymocyte proliferation. The mRNAs for these cytokines are short-lived and are inducible by multiple stimuli. Thus, the steady-state levels for IL-1 and IL-6 mRNAs are critical in establishing the final cytokine protein levels. In this study we have evaluated the effect of epidermal growth factor (EGF), a growth factor for TE cells, and its homologue transforming growth factor alpha (TGF-alpha), on primary cultures of normal human TE cells for the levels of IL-1 alpha, IL-1 beta, IL-6, and TGF-alpha mRNA. We showed that TE cells expressed EGF receptors (EGF-R) in vitro and in vivo, and that treatment of TE cells with EGF or TGF-alpha increased IL-1 and IL-6 biological activity and mRNA levels for IL-1 alpha, IL-1 beta, and IL-6. Neither EGF nor TGF-alpha increased transcription rates of IL-1 alpha, IL-1 beta, and IL-6 genes, but rather both EGF and TGF-alpha increased cytokine mRNA stability. By indirect immunofluorescence assay, TGF-alpha was localized in medullary TE cells and thymic Hassall's bodies while EGF-R was localized to TE cells throughout the thymus. Thus, TGF-alpha and EGF are critical regulatory molecules for production of TE cell-derived cytokines within the thymus and may function as key modulators of human T cell development in vivo.
Localized adherence by enteropathogenic Escherichia coli is an inducible phenotype associated with the expression of new outer membrane proteins.
Authors: Vuopio-Varkila J , Schoolnik G K
Published: June 2004

Enteropathogenic Escherichia coli grow as discrete colonies on the mucous membranes of the small intestine. A similar pattern can be demonstrated in vitro; termed localized adherence (LA), it is characterized by the presence of circumscribed clusters of bacteria attached to the surfaces of cultured epithelial cells. The LA phenotype was studied using B171, an O111:NM enteropathogenic E. coli (EPEC) strain, and HEp-2 cell monolayers. LA could be detected 30-60 min after exposure of HEp-2 cells to B171. However, bacteria transferred from infected HEp-2 cells to fresh monolayers exhibited LA within 15 min, indicating that LA is an inducible phenotype. Induction of the LA phenotype was found to be associated with de novo protein synthesis and changes in the outer membrane proteins, including the production of a new 18.5-kD polypeptide. A partial NH2-terminal amino acid sequence of this polypeptide was obtained and showed it to be identical through residue 12 to the recently described bundle-forming pilus subunit of EPEC. Expression of the 18.5-kD polypeptide required the 57-megadalton enteropathogenic E. coli adherence plasmid previously shown to be required for the LA phenotype in vitro and full virulence in vivo. This observation, the correspondence of the 18.5-kD polypeptide to an EPEC-specific pilus protein, and the temporal correlation of its expression with the development of the LA phenotype suggest that it may contribute to the EPEC colonial mode of growth.
Selection of antigen-specific, idiotype-positive B cells in transgenic mice expressing a rearranged M167-mu heavy chain gene.
Authors: Kenny J J , O'Connell C , Sieckmann D G , Fischer R T , Longo D L
Published: June 2004

Flow cytometric analysis of antigen-specific, idiotype-positive (id+), B cell development in transgenic mice expressing a rearranged M167-mu gene shows that large numbers of phosphocholine (PC)-specific, M167-id+ B cells develop in the spleen and bone marrow of these mice. Random rearrangement of endogenous V kappa genes, in the absence of a subsequent receptor-driven selection, should give rise to equal numbers of T15- and M167-id+ B cells. The observed 100-500-fold amplification of M167-id+ B cells expressing an endogenous encoded V kappa 24]kappa 5 light chain in association with the M167 VH1-id transgene product appears to be an antigen driven, receptor-mediated process, since no amplification of non-PC-binding M167 VH1/V kappa 22, T15-id+ B cells occurs in these mu-only transgenic mice. The selection and amplification of antigen-specific, M167-id+ B cells requires surface expression of the mu transgene product; thus, no enhancement of M167-id+ B cells occurs in the M167 mu delta mem-transgenic mice, which cannot insert the mu transgene product into the B cell membrane. Surprisingly, no selection of PC-specific B cells occurs in M167-kappa-transgenic mice although large numbers of B cells expressing a crossreactive M167-id are present in the spleen and bone marrow of these mice. The failure to develop detectable numbers of M167-id+, PC-specific B cells in M167-kappa-transgenic mice may be due to a very low frequency of M167-VH-region formation during endogenous rearrangement of VH1 to D-JH segments. The somatic generation of the M167 version of a rearranged VH1 gene may occur in less than one of every 10(5) bone marrow B cells, and a 500-fold amplification of this M167-Id+ B cell would not be detectable by flow cytometry even though the anti-PC antibody produced by these B cells is detectable in the serum of M167-kappa-transgenic mice after immunization with PC.
Recombinant Pfs25 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in experimental animals.
Authors: Barr P J , Green K M , Gibson H L , Bathurst I C , Quakyi I A , Kaslow D C
Published: June 2004

Pfs25 is a sexual stage antigen of Plasmodium falciparum that is expressed on the surface of zygote and ookinete forms of the parasite. Monoclonal antibodies directed against native Pfs25 can block completely the development of P. falciparum oocysts in the midgut of the mosquito vector. Thus, this 25-kD protein is a potential vaccine candidate for eliciting transmission-blocking immunity in inhabitants of malaria endemic regions. We have synthesized, by secretion from yeast, a polypeptide analogue of Pfs25 that reacts with conformation-dependent monoclonal antibodies, and elicits transmission-blocking antibodies when used to immunize mice and monkeys in conjunction with a muramyl tripeptide adjuvant. Our results suggest the further evaluation of recombinant DNA-derived Pfs25 in transmission-blocking vaccination studies in humans.
Interleukin 10(IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes.
Authors: de Waal Malefyt R , Abrams J , Bennett B , Figdor C G , de Vries J E
Published: June 2004

In the present study we demonstrate that human monocytes activated by lipopolysaccharides (LPS) were able to produce high levels of interleukin 10 (IL-10), previously designated cytokine synthesis inhibitory factor (CSIF), in a dose dependent fashion. IL-10 was detectable 7 h after activation of the monocytes and maximal levels of IL-10 production were observed after 24-48 h. These kinetics indicated that the production of IL-10 by human monocytes was relatively late as compared to the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF alpha), and granulocyte colony-stimulating factor (G-CSF), which were all secreted at high levels 4-8 h after activation. The production of IL-10 by LPS activated monocytes was, similar to that of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF, inhibited by IL-4. Furthermore we demonstrate here that IL-10, added to monocytes, activated by interferon gamma (IFN-gamma), LPS, or combinations of LPS and IFN-gamma at the onset of the cultures, strongly inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF at the transcriptional level. Viral-IL-10, which has similar biological activities on human cells, also inhibited the production of TNF alpha and GM-CSF by monocytes following LPS activation. Activation of monocytes by LPS in the presence of neutralizing anti-IL-10 monoclonal antibodies resulted in the production of higher amounts of cytokines relative to LPS treatment alone, indicating that endogenously produced IL-10 inhibited the production of IL-1 alpha, IL-1 beta, IL-6, IL-8, TNF alpha, GM-CSF, and G-CSF. In addition, IL-10 had autoregulatory effects since it strongly inhibited IL-10 mRNA synthesis in LPS activated monocytes. Furthermore, endogenously produced IL-10 was found to be responsible for the reduction in class II major histocompatibility complex (MHC) expression following activation of monocytes with LPS. Taken together our results indicate that IL-10 has important regulatory effects on immunological and inflammatory responses because of its capacity to downregulate class II MHC expression and to inhibit the production of proinflammatory cytokines by monocytes.
The cytoplasmic domain of the integrin lymphocyte function-associated antigen 1 beta subunit: sites required for binding to intercellular adhesion molecule 1 and the phorbol ester-stimulated phosphorylation site.
Authors: Hibbs M L , Jakes S , Stacker S A , Wallace R W , Springer T A
Published: June 2004

We have defined the regions of the cytoplasmic domain of the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1) that are required for active binding of its extracellular domain to intercellular adhesion molecule 1 (ICAM-1). The NH2-terminal 28 amino acids in the cytoplasmic domain are dispensable, but a segment of 5 amino acids including three contiguous threonines (758-760) and Phe 766 in the COOH-terminal third of the cytoplasmic domain are required for binding to ICAM-1. Mutation and phosphoamino acid analysis show that Ser 756 is the major residue phosphorylated in response to phorbol ester. Furthermore, multiple mutations demonstrate that serine phosphorylation can be dissociated from phorbol ester-stimulated binding of LFA-1 to ICAM-1. The sites we have defined are previously unremarked, are well conserved in the beta 1, beta 3, and beta 7 integrin subunits, and may be of broad importance in regulating adhesiveness of integrins.
Purification and characterization of a novel soluble receptor for interleukin 1.
Authors: Symons J A , Eastgate J A , Duff G W
Published: June 2004

Affinity chromatography and reverse-phase high-performance liquid chromatography was used to purify a soluble interleukin 1 beta (IL-1 beta) specific binding protein from the supernatant of a human B cell line, Raji. The purified protein specifically bound 125I IL-1 beta forming a 60-kD complex in nonreducing conditions and a 70-kD complex in reducing conditions. Binding was found to be displaceable by mature human and murine IL-1 beta and human 31-kD IL-1 beta propeptide, but not displaceable by human and murine IL-1 alpha or human IL-1 receptor (IL-1R) antagonist. Ligand blotting revealed a 47-kD molecule that specifically bound IL-1 beta. Measurement of binding affinity of the cell surface Raji IL-1R (Kd = 2.2 nm) and the Raji soluble (s)IL-1R (Kd = 2.7 nm) demonstrated a similar affinity for 125I IL-1 beta. Purified sIL-1R inhibited binding of IL-1 beta to cell lines with both type I (80 kD) and type II (65 kD) IL-1Rs, but did not interfere with IL-1 alpha binding. This natural sIL-1R may function as an important regulatory molecule of IL-1 beta in vivo.
Differential regulation of transforming growth factor beta and interleukin 2 genes in human T cells: demonstration by usage of novel competitor DNA constructs in the quantitative polymerase chain reaction.
Authors: Li B , Sehajpal P K , Khanna A , Vlassara H , Cerami A , Stenzel K H , Suthanthiran M
Published: June 2004

The regulation of mRNA encoding transforming growth factor beta (TGF-beta) and interleukin 2 (IL-2) in normal human T cells was explored using novel competitor DNA constructs in the quantitative polymerase chain reaction and accessory cell-independent T cell activation models. Our experimental design revealed the following: (a) TGF-beta mRNA and IL-2 mRNA are regulated differentially in normal human T cells, quiescent or signaled with the synergistic combinations of: sn-1,2-dioctanoylglycerol and ionomycin or anti-CD3 monoclonal antibody (mAb) and anti-CD2 mAb; (b) the steady-state level of TGF-beta mRNA in the stimulated T cells, in contrast to that of IL-2 mRNA, is increased by the immunosuppressant cyclosporine (CsA); and (c) the paradoxical effect of CsA on TGF-beta mRNA levels is also appreciable at the level of production of functionally active TGF-beta protein. Our findings, in addition to demonstrating the utility of the competitor DNA constructs for the precise quantification of immunoregulatory cytokines, suggest a novel and unifying mechanistic basis for the immunosuppression and some of the complications (e.g., renal fibrosis) associated with CsA usage.
Characterization and clonal distribution of four alleles of the speA gene encoding pyrogenic exotoxin A (scarlet fever toxin) in Streptococcus pyogenes.
Authors: Nelson K , Schlievert P M , Selander R K , Musser J M
Published: June 2004

Streptococcus pyogenes strains producing pyrogenic exotoxin A (scarlet fever toxin) have recently caused episodes of streptococcal toxic-shock-like syndrome (TSLS). We exploited knowledge of genetic diversity and relationships among exotoxin A-producing patient strains provided by multilocus enzyme electrophoresis to select strains for comparative sequencing of toxin genes. Our analysis identified four alleles of speA in natural populations, one of which (speA1) occurs in many distinct clonal lineages and is probably old. Two other alleles (speA2 and speA3), characterized solely by single amino acid substitutions, were each identified in single clones that together have caused the majority of TSLS episodes. It is unlikely that these alleles have had a long association with S. pyogenes clones. A fourth allele (speA4) also is present in a single phylogenetic lineage and is 9% divergent from the other three toxin alleles. An absence of synonomous (silent) nucleotide changes in speA2 and speA3 is unusual and suggests that the allelic variation is not selectively neutral, which implies that the toxins are not functionally equivalent. These results may be important in helping to understand the recent increase in frequency and severity of disease caused by S. pyogenes.
The junctional modifications of a T cell receptor gamma chain are determined at the level of thymic precursors.
Authors: Ikuta K , Weissman I L
Published: June 2004

T precursors from fetal liver and adult bone marrow were compared for their ability to give rise to V gamma 4+ T cell development. Fetal thymic lobes were repopulated with fetal liver or adult bone marrow cells, and the organ-cultured thymocytes were analyzed for their T cell receptor expression by the polymerase chain reaction (PCR). Both day 14 fetal liver and adult bone marrow cells gave rise to thymocytes with V gamma 4-J gamma 1 transcripts. However, the average size of the PCR products derived from adult precursors was slightly larger than that from fetal precursors. DNA sequence analysis of the V gamma 4-J gamma 1 transcripts showed that early fetal liver precursors predominantly gave rise to thymocytes with the V gamma 4-J gamma 1 transcripts without N nucleotide insertion, while late fetal liver and adult marrow precursors predominantly gave rise to thymocytes with modified V gamma 4-J gamma 1 junctions. These results suggest the possibility that the level of the N nucleotide insertion is programmed at the level of thymic precursors. This study also supported the model presented previously that the developmental potential of hematopoietic stem cells may change during ontogeny.
Anti-CD2 antibodies induce T cell unresponsiveness in vivo.
Authors: Gückel B , Berek C , Lutz M , Altevogt P , Schirrmacher V , Kyewski B A
Published: June 2004

The CD2 receptor functions as an adhesion and signal molecule in T cell recognition. Multimeric binding of CD2 on T cells to its physiologic ligand LFA-3 on cognate partner cells in vitro efficiently augments the antigen-specific T cell signal delivered by the T cell receptor/CD3 complex. The precise contribution of the antigen-nonspecific CD2-LFA-3 interactions to T cell immune responses in vivo, however, has been difficult to assess. Here we analyzed the role of CD2 in the murine immune response using a nondepleting anti-CD2 monoclonal antibody that induces a marked, reversible modulation of CD2 expression on murine T and B cells in situ. This modulation is dose and time dependent, specific for CD2, and does not require the Fc portion of the antibody. Anti-CD2 antibodies [rat IgG1 or F(ab')2] significantly inhibit the CD4+ T cell-mediated response to hen egg lysozyme and the cytotoxic CD8+ T cell response to a syngeneic tumor cell line. In both cases, anti-CD2 antibodies are only effective when administered before or within 24 h after antigen priming. The suppression of the antitumor response corresponds to a sixfold reduction of specific cytotoxic T lymphocyte precursor cells and results in the abrogation of protective antitumor immunity. Anti-CD2 antibodies also affect the humoral immune response to oxazolone: the isotype switch from specific IgM to IgG1 antibodies is delayed, whereas the IgM response is unaltered. In addition, a single antibody injection results in sustained polyclonal unresponsiveness of T cells irrespective of antigen priming and CD2 modulation. These results document that CD2-mediated signals induce a state of T cell unresponsiveness in vivo.
T cell memory is short-lived in the absence of antigen.
Authors: Gray D , Matzinger P
Published: June 2004

Immunological memory has generally been ascribed to the development of long-lived memory cells that can persist for years in the absence of renewed antigenic encounter. In the experiments reported here, we have adoptively transferred memory T cells in the presence and absence of priming antigen and assessed their functional survival. The results indicate that, in contrast to the traditional view, the maintenance of T cell memory requires the presence of antigen, suggesting that memory, like tolerance, is an antigen-dependent process rather than an antigen-independent state.
The V beta 17+ T cell repertoire: skewed J beta usage after thymic selection; dissimilar CDR3s in CD4+ versus CD8+ cells.
Authors: Candéias S , Waltzinger C , Benoist C , Mathis D
Published: June 2004

To ascertain how the actual repertoire of T cell receptors (TCRs) deviates from the theoretical, we have generated a large number of junctional region sequences from TCRs carrying the V beta 17 variable region. The greater than 600 sequences analyzed represent transcripts from nine different cell populations, permitting several comparisons: transcripts from an expressed vs. a non-expressed V beta 17 allele, those from E+ vs. E- mice, transcripts from immature vs. mature thymocytes, those from thymic vs. peripheral T cells, and those from CD4+ vs. CD8+ cells. These comparisons have allowed us to distinguish between the influence of molecular events involved in TCR gene rearrangement and that of various selection events that shape the T cell repertoire. Our most striking findings are: (a) that J beta usage is markedly skewed, partly due to recombination mechanics and partly due to selection forces: in particular, those mediated by the class II E molecule in the thymus; and (b) that TCRs on CD4+ and CD8+ cells show intriguing dissimilarities. In addition, we present evidence that N nucleotide additions occur with clear biases, probably due to idiosyncrasies of the recombination enzymes, and provide arguments that TCR and immunoglobulin CDR3s have distinct structures.
Interleukin 7 induces CD4+ T cell-dependent tumor rejection.
Authors: Hock H , Dorsch M , Diamantstein T , Blankenstein T
Published: June 2004

The potential of interleukin 7 (IL-7) to induce an antitumor response in vivo was analyzed. Therefore, the IL-7 gene was expressed in the plasmacytoma cell line J558L. Although the growth of IL-7-producing cells was not retarded in vitro, the IL-7-producing cells were completely rejected upon injection into mice. Tumor rejection was observed only in syngeneic but not in nude mice. The tumor-suppressive effect could be abolished by the parallel injection of an anti-IL-7 monoclonal antibody. Immunohistochemical analysis revealed IL-7-dependent infiltration of the tumor tissue by CD4+ and CD8+ T lymphocytes, and also type 3 complement receptor-positive (CR3+) cells, predominantly macrophages. Depletion of T cell subsets in tumor-bearing mice showed the absolute dependence of the antitumor response on CD4+ cells, whereas tumor rejection was unaffected by depletion of CD8+ cells. In addition to CD4+ cells, CR3+ cells were also needed for tumor rejection. The antitumor effect of IL-7 was confirmed by expression of the IL-7 gene in a second tumor cell line of different cellular origin. Together, our results demonstrate that a high local IL-7 concentration at the tumor site obtained by tumor cell-targeted gene transfer leads to tumor rejection involving a cellular mechanism that seems to be different from the ones observed in analogous experiments with other cytokines.
Determination of the structural basis for selective binding of Epstein-Barr virus to human complement receptor type 2.
Authors: Martin D R , Yuryev A , Kalli K R , Fearon D T , Ahearn J M
Published: June 2004

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that selectively infects and immortalizes human B lymphocytes. One determinant of this narrow tropism is human CR2, the only viral receptor within the superfamily of proteins that contain short consensus repeats (SCRs). Human CR2 serves as a receptor for both C3dg and the gp350/220 glycoprotein of EBV, and binds the monoclonal antibody (mAb) OKB7, which blocks binding of both ligands to the receptor. In contrast, although murine CR2 is capable of binding human C3dg and this interaction can be blocked with the mAb 7G6, it does not bind OKB7 or EBV. We have determined the structural basis for absolute specificity of EBV for human CR2 through characterization of a panel of 24 human-murine chimeric receptors, all of which bind human C3dg. The results indicate that preferential binding of EBV to human CR2 is not due to unique amino acids that are capable of binding the virus, but reflects a distinct receptor conformation that can be achieved in murine CR2 with single amino acid substitutions in two discontinuous regions of the primary structure: replacement of proline at position 15 with the corresponding serine from human CR2, and elimination of a potential N-linked glycosylation site between SCR-1 and SCR-2. Furthermore, species-specific binding of EBV, OKB7, and 7G6 can all be manipulated through substitutions among residues 8-15, suggesting that this octapeptide is part of a structural determinant that is critical for binding of both viral and natural ligands to CR2.
Regulated expression and function of CD11c/CD18 integrin on human B lymphocytes. Relation between attachment to fibrinogen and triggering of proliferation through CD11c/CD18.
Authors: Postigo A A , Corbí A L , Sánchez-Madrid F , de Landázuri M O
Published: June 2004

CD11c/CD18 (p150,95) is a beta 2 integrin expressed by myeloid, natural killer and certain lymphoid cells such as some cytotoxic T cell clones and B cell malignancies. We have studied the expression and function of CD11c on resting and activated B lymphocytes. Flow cytometry, immunoprecipitation, and mRNA analyses showed that cell activation with phorbol esters or with a variety of stimuli such as Staphylococcus aureus or anti-mu antibodies in combination with cytokines induced de novo CD11c/CD18 cell surface expression on most B cells while CD11b expression was not affected. Functional analysis of CD11c/CD18 on B cells revealed that it plays a dual role. First, CD11c/CD18 is implicated in B cell proliferation, as demonstrated by the ability of several anti-CD11c monoclonal antibodies to trigger comitogenic signals; and second, the newly expressed CD11c/CD18 mediates B cell binding to fibrinogen. Our data conclusively demonstrate the role of CD11c/CD18 on both B cell activation and adhesion processes.
Platelet-derived growth factor is a potent biologic response modifier of T cells.
Authors: Daynes R A , Dowell T , Araneo B A
Published: June 2004

Freshly isolated lymph node (LN) cells cultured in serum-containing medium were restricted to produce primarily interleukin 2 (IL-2) subsequent to T cell activation. Only minimal amounts of IL-4, IL-5, or interferon gamma (IFN-gamma) were produced under these conditions. Similar populations of LN cells cultured in serum-free medium were able to produce a variety of lymphokines after T cell activation, with the relative quantities of each species being dependent upon the lymphoid organ source of the lymphocytes. A similar relationship in the patterns of lymphokines produced by activated T cell hybridomas maintained under serum-free conditions was also observed, whereas activation in serum-supplemented media resulted in a predominant restriction to the secretion of IL-2. Additional studies determined that the entity in serum responsible for restricting T cell function in vitro was platelet-derived growth factor (PDGF). The PDGF-BB isoform was established to be the most active in the regulation of T cell function, enhancing IL-2 while depressing the production of IL-4, IL-5, and IFN-gamma at concentrations below 1 ng/ml. PDGF-AB was also found to be quite active, however, this isoform of PDGF was incapable of influencing IFN-gamma production at the concentrations tested. PDGF-AA was very weakly active. It therefore appears that PDGF, acting primarily through a beta receptor subunit (either alpha/beta- or beta/beta-type receptors) is able to influence profoundly the behavior of T cells, with some of its modulatory effects exhibiting isoform specificity. This is reflected by an enhancement in the production of IL-2, while simultaneously depressing the secretion of IL-4, IL-5, and IFN-gamma (PDGF-BB only) after T cell activation. Kinetic studies, where cell supernatants were analyzed both 24 and 48 h after T cell activation, suggested that "desensitization" to PDGF influences can occur naturally in vitro. Those species of lymphokines that were inhibited by PDGF over the first 24 h after activation could be produced at normal levels over the subsequent 24-h period. Finally, lymphokines maintained in the presence of PDGF-BB for greater than 24 h before their activation lost sensitivity to this growth factor. These cells regained responsiveness to PDGF after an additional incubation period in PDGF-free medium. Collectively, our data imply that the pattern of T cell lymphokines produced, plus the kinetics of their production after activation, are being controlled by the potent serum growth factor PDGF.(ABSTRACT TRUNCATED AT 400 WORDS)
B cell tolerance in mice transgenic for anti-CD8 immunoglobulin mu chain.
Authors: Brombacher F , Köhler G , Eibel H
Published: June 2004

To analyze in vivo the induction of B cell tolerance against a T cell surface antigen, we generated transgenic mice expressing an anti-CD8.2 mu heavy chain gene. We show that self-specific B cells are efficiently tolerized if they express the membrane-bound form of the transgenic mu chain on their surface but that they can escape tolerization if they express only the secreted form. In the latter, we find an enhanced expression of anti-CD8.2 antibodies after polyclonal B cell activation. As a result, transgenic anti-CD8.2 antibodies bind to the CD8+ T cells but they did not induce their elimination. Furthermore, we observed the preferential expression of a limited subset of endogenous light chains with the transgenic mu chain. This suggests a positive or negative selection for particular heavy and light chain combinations in B lymphocytes.
Identification of the motility-related protein (MRP-1), recognized by monoclonal antibody M31-15, which inhibits cell motility.
Authors: Miyake M , Koyama M , Seno M , Ikeyama S
Published: June 2004

A murine monoclonal antibody (M31-15) was identified using the penetration-inhibiting assay of a human lung adenocarcinoma cell line (MAC10) and remarkably inhibited the phagokinetic tract motility of various cancer cell lines. The antigen, motility-related protein (MRP-1), recognized by M31-15, was 25- and 28-kD proteins, and M31-15 was used to isolate a cDNA clone from a human breast carcinoma cDNA library. Sequence analysis revealed that MRP-1 had strong similarity with a B cell surface antigen (CD37), a melanoma-associated antigen (ME491), the target of an antiproliferative antibody (TAPA-1), a human tumor-associated antigen (CO-029), and the Sm23 antigen of the trematode parasite Schistosoma mansoni.
Sphingosine synergistically stimulates tumor necrosis factor alpha-induced prostaglandin E2 production in human fibroblasts.
Authors: Candela M , Barker S C , Ballou L R
Published: June 2004

Sphingosine is a biologically active derivative of sphingomyelin. It affects diverse cellular functions and its mechanism(s) of action is poorly defined. Tumor necrosis factor alpha (TNF alpha) has recently been shown to rapidly induce sphingomyelin turnover, implicating this metabolic pathway in TNF alpha signal transduction. Because TNF alpha is known to induce prostaglandin E2 (PGE2) production in human fibroblasts, we tested the effect of sphingosine on TNF alpha-induced PGE2 production. We found that sphingosine enhanced TNF alpha-induced PGE2 production by as much as 18-fold over TNF alpha alone. Sphingosine appeared to stimulate TNF alpha-induced PGE2 production independent of TNF alpha-mediated interleukin 1 (IL-1) production, because anti-IL-1 antibodies and IL-1 receptor antagonist protein (IRAP) did not inhibit TNF alpha-induced PGE2 production or the stimulatory effect of sphingosine. TNF alpha stimulated PGE2 production to the same degree in normal and protein kinase C (PKC) downregulated cells in the presence and absence of sphingosine, indicating that neither TNF alpha nor sphingosine require active PKC to elicit their respective effects. The sphingosine analogues stearylamine and stearoyl-D-sphingosine had little or no effect on TNF alpha-mediated PGE2 production, supporting a specific role for sphingosine in the activation process. Short-term (1 min) exposure of cells to sphingosine dramatically increased TNF alpha-induced PGE2 production. A potential mechanism by which sphingosine could increase TNF alpha-induced PGE2 production involves enhancement of phospholipase A2 (PLA2) and/or cyclooxygenase (Cox) activity, the rate-limiting enzymes in PGE2 production. We found that both TNF alpha and sphingosine alone enhanced these enzymatic activities, and that sphingosine additively increased the effect of TNF alpha on phospholipase A2 activity. It appears that sphingosine affects TNF alpha-induced PGE2 production via a mechanism that is independent of PKC involvement, and that sphingosine may function as an endogenous second messenger capable of modulating the responsiveness of the cell to external stimuli.
T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire.
Authors: Casanova J L , Romero P , Widmann C , Kourilsky P , Maryanski J L
Published: June 2004

We report here the first extensive study of a T cell repertoire for a class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocyte (CTL) response. We have found that the T cell receptors (TCRs) carried by 28 H-2Kd-restricted CTL clones specific for a single Plasmodium berghei circumsporozoite nonapeptide are highly diverse in terms of V alpha, J alpha, and J beta segments and aminoacid composition of the junctional regions. However, despite this extensive diversity, a high proportion of the TCRs contain the same V beta segment. These results are in contrast to most previously reported T cell responses towards class II MHC-peptide complexes, where the TCR repertoires appeared to be much more limited. In our study, the finding of a dominant V beta in the midst of otherwise highly diverse TCRs suggests the importance of the V beta segment in shaping the T cell repertoire specific for a given MHC-peptide complex. As an additional finding, we observed that nearly all clones have rearranged both TCR alpha loci. Moreover, as many as one-third of the CTL clones that we analyzed apparently display two productive alpha rearrangements. This argues against a regulated model of sequential recombination at the alpha locus and consequently raises the question of whether allelic exclusion of the TCR alpha chain is achieved at all.
Hemolysis of human erythrocytes is a new bioactivity of gangliosides.
Authors: Horikawa K , Nakakuma H , Nagakura S , Kawakita M , Kagimoto T , Iwamori M , Nagai Y , Abe T , Takatsuki K
Published: June 2004

Using sheep erythrocytes and liposomes, an inhibitory effect of gangliosides has been shown on the activation of the alternative pathway of complement. However, in studies using human erythrocytes, we found that gangliosides had hemolytic activity that was possibly mediated through activation of the alternative pathway. Pretreatment of human erythrocytes obtained from healthy volunteers or paroxysmal nocturnal hemoglobinuria (PNH) patients with a ganglioside mixture purified from human erythrocytes enhanced their susceptibility to homologous human complement, and resulted in dose-dependent hemolysis. The enhancement was more marked in PNH erythrocytes than control cells. Protease treatment of the ganglioside mixture did not change its hemolytic activity, but sialidase treatment abolished the activity. Among the major erythrocyte gangliosides, II3NeuAc-LacCer (GM3) was the most potent hemolytic agent. Gangliosides purified from bovine brain were also active, while neither nonsialylated glycosphingolipids, the ceramide moiety, or sialic acid alone were active. Sialic acid residues in the ganglioside molecules were essential to this activity, but the amount of the residue or the source of the gangliosides seemed not to be important. Several treatments inhibiting the alternative but not classical complement pathway markedly reduced the ganglioside hemolytic activity. This novel bioactivity of gangliosides was thus suggested to be mediated partly by activation of the alternative pathway.
CD69-mediated pathway of lymphocyte activation: anti-CD69 monoclonal antibodies trigger the cytolytic activity of different lymphoid effector cells with the exception of cytolytic T lymphocytes expressing T cell receptor alpha/beta.
Authors: Moretta A , Poggi A , Pende D , Tripodi G , Orengo A M , Pella N , Augugliaro R , Bottino C , Ciccone E , Moretta L
Published: June 2004

The effect of anti-CD69 monoclonal antibodies (mAbs) on the induction of the cytolytic activity in different types of lymphoid effector cells has been investigated. Three anti-CD69 mAbs, including the reference mAb MLR3 and two new mAbs (c227 and 31C4), have been used. All cloned CD3-CD16+ natural killer (NK) cells belonging to different subsets (as defined by the surface expression of GL183 and/or EB6 antigens) were efficiently triggered by anti-CD69 mAbs and lysed P815 mastocytoma cells in a redirected killing assay. Triggering of the cytolytic activity could also be induced in CD3-CD16- NK clones, which fail to respond to other stimuli (including anti-CD16, anti-CD2 mAbs, or phytohemagglutinin). A similar triggering effect was detected in T cell receptor (TCR) gamma/delta+ clones belonging to different subsets. On the other hand, anti-CD69 mAbs could not induce triggering of the cytolytic activity in TCR alpha/beta+ cytolytic clones. Since all thymocytes are known to express CD69 antigen after cell activation, we analyzed a series of phenotypically different cytolytic thymocyte populations and clones for their responsiveness to anti-CD69 mAb in a redirected killing assay. Again, anti-CD69 mAb triggered TCR gamma/delta+ but not TCR alpha/beta+ thymocytes. Anti-CD69 mAb efficiently triggered the cytolytic activity of "early" thymocytes lines or clones (CD3-4-8-7+), which lack all other known pathways of cell activation. Thus, it appears that CD69 molecules may initiate a pathway of activation of cytolytic functions common to a number of activated effector lymphocytes with the remarkable exception of TCR alpha/beta+ cytolytic cells.
Human immunodeficiency virus type 1 activates the classical pathway of complement by direct C1 binding through specific sites in the transmembrane glycoprotein gp41.
Authors: Ebenbichler C F , Thielens N M , Vornhagen R , Marschang P , Arlaud G J , Dierich M P
Published: June 2004

Human immunodeficiency virus type 1 (HIV-1), in contrast to animal retroviruses such as murine leukemia virus, is not lysed by human complement. Nevertheless, HIV-1 activates complement via the classical pathway independent of antibody, and C3b deposition facilitates infection of complement receptor-bearing cells. Using gel exclusion chromatography on Sephacryl S-1000, purified virions were found to bind 125I-labeled C1q, but not 125I-labeled dimeric proenzyme C1s. Virions activated the C1 complex, reconstituted from C1q, proenzyme C1r, and 125I-labeled proenzyme C1s, to an extent comparable with that obtained with immunoglobulin G-ovalbumin immune complexes. To determine the activating viral component, recombinant viral proteins were used: in the solid phase, soluble gp41 (sgp41) (the outer membrane part of gp41, residues 539-684 of gp160) bound C1q, but not dimeric proenzyme C1s, while gp120 was ineffective. In the fluid phase, sgp41 activated the C1 complex in a dose- and time-dependent manner, more efficiently than aggregated Ig, but less efficiently than immune complexes. To localize the C1 activating site(s) in gp41, synthetic peptides (15-residue oligomers spanning amino acids 531-695 of gp160) were used. Peptides covering positions 591-605 and 601-620 and, to a lesser extent, positions 561-575, had both the ability to bind C1q and to induce C3 deposition. These data provide the first experimental evidence of a direct interaction between the C1 complex and HIV-1, and indicate that C1 binding and activation are mediated by specific sites in gp41.
Reduction of lupus nephritis in MRL/lpr mice by a bacterial superantigen treatment.
Authors: Kim C , Siminovitch K A , Ochi A
Published: June 2004

The effects of biweekly intravenous injections of Staphylococcus Enterotoxin B (SEB) into autoimmune MRL-lpr/lpr (MRL/lpr) mice were investigated. Rather than causing the expansion of V beta 8+ T cells, SEB administration resulted in the reduction V beta 8+, CD4-CD8- "double-negative" (DN) T cells. This was shown by FACS analysis as this putative pathogenic population was diminished in both spleen and lymph node. The symptoms of systemic lupus erythematosus (SLE) in MRL/lpr, which include high titers of anti-DNA antibodies and circulating immune complexes and proteinuria, were reduced in SEB-treated mice in a dose-dependent manner. The clinical parameters of SLE in MRL/lpr, which include lymph node hyperplasia and necrotic vasculitis, were suppressed in 50-micrograms SEB-treated mice. T cells bearing V beta 6 T cell receptor, which does not interact with SEB, were not reduced with SEB administration. Thus, disease suppression was associated with a specific reduction in the number of V beta 8+, DN T cells. These results implicate a possible therapeutic role of superantigen-based immunotherapy in V beta-restricted, T cell-dominated clinical syndromes.
The cutaneous lymphocyte antigen is a skin lymphocyte homing receptor for the vascular lectin endothelial cell-leukocyte adhesion molecule 1.
Authors: Berg E L , Yoshino T , Rott L S , Robinson M K , Warnock R A , Kishimoto T K , Picker L J , Butcher E C
Published: June 2004

A skin-associated population of memory T lymphocytes, defined by expression of the cutaneous lymphocyte antigen (CLA), binds selectively and avidly to the vascular lectin endothelial cell-leukocyte adhesion molecule 1 (ELAM-1), an interaction that may be involved in targeting of CLA+ T cells to cutaneous sites of chronic inflammation. Here we present evidence that CLA itself is the (or a) lymphocyte homing receptor for ELAM-1. Antigen isolated with anti-CLA monoclonal antibody HECA-452 from human tonsillar lysates avidly binds ELAM-1 transfected mouse cells. Anti-CLA antibody blocks T lymphocyte binding to ELAM-1 transfectants. HECA-452 and ELAM-1 binding to lymphocytes or to isolated tonsillar HECA-452 antigen is abrogated by neuraminidase treatment implying a prominent role for sialic acid in CLA structure and function. The dominant form of CLA on T cells is immunologically distinct from the major neutrophil ELAM-1 ligand, the sialyl Lewis x (sLex) antigen (NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc), which is absent, weakly expressed, or masked on T cells. However, neuraminidase treatment of CLA+ T cells, but not of CLA- T cells, reveals Lewis x (CD15) structures. In combination with the known requirement for terminal NeuAc alpha 2-3Gal and fucose residues attached to N-acetylglucosamine for ELAM-1 and HECA-452 binding, this finding suggests that CLA may comprise an additionally sialylated or otherwise modified form of sLex. The identification of a lymphocyte homing receptor for skin may permit novel approaches to the diagnosis and therapy of cutaneous and inflammatory disorders.
Analysis of T cell receptor beta chains in Lewis rats with experimental allergic encephalomyelitis: conserved complementarity determining region 3.
Authors: Gold D P , Offner H , Sun D , Wiley S , Vandenbark A A , Wilson D B
Published: June 2004

This study explores the usage of T cell antigen receptor (TCR) beta chain elements in Lewis rats with experimentally induced allergic encephalomyelitis (EAE). TCRs from 15 different T cell clones and hybridomas derived from animals immunized with myelin basic protein (MBP), and all having specificity for the 21-mer encephalitogenic fragment MBP 68-88, utilized V beta 8.2. In addition, there was a marked conservation of the first two amino acid residues of the junctional complementarity determining region 3 (CDR3) associated with the V beta 8.2 receptors. 12 of 15 contained an aspartic acid followed by serine regardless of the associated J beta element. At the nucleotide level, this conservation of AspSer residues was accomplished with few or no nongermline-encoded nucleotide (N) additions. A similar pattern of AspSer usage and N region nucleotide additions was observed in a number of V beta 8.2 isolates derived from MBP-immunized lymph nodes. In contrast, V beta 8.2 polymerase chain reaction amplified isolates from Lewis T cells activated with concanavalin A or from lymph nodes of complete Freund's adjuvant-immunized animals showed no AspSer utilization (0/31) in the CDR3, and four to nine N region nucleotide additions. We conclude from this finding that AspSer residues in the CDR3, limited N region nucleotide additions, along with V beta 8.2 sequences, contribute to TCR specificity for MBP 68-88. This raises the possibility that encephalitogenic, disease-causing T cells either represent a population that derives from late fetal life or alternatively, that they are rare cells with this particular TCR phenotype contributed to the T cell pool throughout adulthood and are selected by antigen. In either case, the CDR3 AspSer sequences as well as V beta 8.2 sequences are candidates for the receptor target structures recognized by regulator T cells in recovery from and resistance to active EAE. In this respect, a preliminary analysis of TCR utilization in three T cell clones specific for MBP 68-88 isolated from animals recovered from active EAE indicates that while all three use V beta 8.2, only one contains AspSer in the CDR3.
Productive human immunodeficiency virus type 1 (HIV-1) infection of nonproliferating human monocytes.
Authors: Weinberg J B , Matthews T J , Cullen B R , Malim M H
Published: June 2004

Human immunodeficiency virus type 1 (HIV-1) infection of T lymphocytes requires cellular proliferation and DNA synthesis. Human monocytes were shown to have low DNA synthesis rates, yet the monocytotropic BaL isolate of HIV-1 was able to infect these cells efficiently. Monocytes that were irradiated to assure no DNA synthesis could also be readily infected with HIV-1BaL. Such infections were associated with the integration of HIV-1BaL DNA into the high molecular weight, chromosomal DNA of monocytes. Thus, normal, nonproliferating monocytes differ from T lymphocytes in that a productive HIV-1 infection can occur independently of cellular DNA synthesis. These results suggest that normal nonproliferating mononuclear phagocytes, which are relatively resistant to the destructive effects of this virus, may serve as persistent and productive reservoirs for HIV-1 in vivo.
A tumor necrosis factor (TNF) receptor-IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.
Authors: Peppel K , Crawford D , Beutler B
Published: June 2004

Using a multistep polymerase chain reaction method, we have produced a construct in which a cDNA sequence encoding the extracellular domain of the human 55-kD tumor necrosis factor (TNF) receptor is attached to a sequence encoding the Fc portion and hinge region of a mouse IgG1 heavy chain through an oligomer encoding a thrombin-sensitive peptide linker. This construct was placed downstream from a cytomegalovirus promoter sequence, and expressed in Chinese hamster ovary cells. A secreted protein, capable of binding TNF and inactivating it, was produced by the transfected cells. Molecular characterization revealed that this soluble version of the TNF receptor was dimeric. Moreover, the protein could be quantitatively cleaved by treatment with thrombin. However, the monovalent extracellular domain prepared in this way has a greatly reduced TNF inhibitory activity compared with that of the bivalent inhibitor. Perhaps because of its high affinity for TNF, the chimeric protein is far more effective as a TNF inhibitor than are neutralizing monoclonal antibodies. This molecule may prove very useful as a reagent for the antagonism and assay of TNF and lymphotoxin from diverse species in health and disease, and as a means of deciphering the exact mechanism through which TNF interacts with the 55-kD receptor.