bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelial cells augment the expression of CD40 ligand on newly activated human CD4+ T cells through a CD2/LFA‐3 signaling pathway
Authors: Karmann Karin , Hughes Christopher C. W. , Fanslow William C. , Pober Jordan S.
Published: March 2007

Cultured human endothelial cells (EC) increase CD40 ligand expression on polyclonally activated human peripheral blood CD4+ helper T cells compared to T cells activated in the absence of accessory cells or in the presence of peripheral blood adherent cells or B cells. Induction of CD40 ligand expression appears to be biphasic with early induction observable at 6 h and later induction observable at 24 h. EC cause T cells to increase CD40 ligand expression during the early phase at 6 h after activation. CD40 ligand expression is restricted to the CD4+ helper T cell subset of the peripheral blood T cells, even when EC are present. Blocking monoclonal antibodies to co‐stimulatory molecules on EC and T cells indicate that the CD2/LFA‐3 pathway, which also contributes to induction of augmented interleukin‐2 (IL‐2) secretion, is involved in EC‐induced upregulation of CD40 ligand. Exogenous IL‐2 can also increase CD40 ligand expression. However, increased IL‐2 secretion in the presence of EC can not fully account for endothelial‐induced CD40 ligand up‐regulation as (1) the effect of exogenous IL‐2 is greater at 24 h than at 6 h, whereas the opposite is true for EC; (2) the effect of saturating levels of IL‐2 is considerably smaller than that of EC; and (3) blocking of IL‐2 receptors does not fully inhibit endothelial effects on CD40 ligand expression. We conclude that EC provide unique co‐stimulatory signals that affect the phenotype of activated CD4+ T cells.
Interleukin‐12 is produced by dendritic cells and mediates T helper 1 development as well as interferon‐γ production by T helper 1 cells
Authors: Heufler Christine , Koch Franz , Stanzl Ursula , Topar Gerda , Wysocka Maria , Trinchieri Giorgio , Enk Alexander , Steinman Ralph M. , Romani Nikolaus , Schuler Gerold
Published: March 2007

Interleukin‐12 (IL‐12), a 70‐kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon‐γ (IFN‐γ), low IL‐4]. Established sources of IL‐12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell‐mediated immunity, we reasoned that DC should constitute a critical source of IL‐12. The criteria used to detect IL‐12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, Northern blotting, and in situ hybridization) as well as IL‐12 protein (p70 enzyme‐linked immunosorbent assay, p70 antigen capture followed by IFN‐γ bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL‐12 bymurine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral blood mononuclear cells. DC exhibited, however, features that had not been seen with other antigen‐presenting cells: they produced bioactive IL‐12 upon antigen‐specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti‐IL‐12 antibodies showed that DC‐derived IL‐12 was critical for optimal proliferation and IFN‐γ production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC‐derived IL‐12, but did not require anti‐IL‐4, exogenous IL‐12, or microbes, might be a major function of DC.
Abnormally short serum half‐lives of IgG in β2‐microglobulin‐deficient mice
Authors: Ghetie Victor , Hubbard James G. , Kim Jin‐Kyoo , Tsen May‐Fang , Lee Yukfung , Ward E. Sally
Published: March 2007

The MHC class I‐related receptor, FcRn, mediates the transfer of maternal gamma globulin (IgG) to young rodents, primarily via intestinal transcytosis, and this provides humoral immunity for the first few weeks after birth. In a previous study, the site of mouse IgG1 (mIgG1) with which FcRn interacts has been mapped using recombinant wild‐type and mutated Fc‐hinge fragments. The site encompasses residues at the CH2‐CH3 domain interface of Fc (Ile253, His310, Gln311, His433 and Asn434) and the same amino acids are involved in regulating the pharmacokinetics of the Fc‐hinge fragments. This suggests that in addition to its known function, FcRn might also play a role in IgG homeostasis. Consistent with this hypothesis, in this study, we demonstrate that FcRn α‐chain mRNA is present not only in neonatal brush border but also in other tissues of adult animals (liver, lung, spleen and endothelial cells). In addition, analysis of the pharmacokinetics of mouse Ig/Fc‐hinge fragments in genetically manipulated mice that are deficient in the expression of FcRn demonstrates that the β‐phase half‐lives are abnormally short. These findings suggest that FcRn is involved in IgG homeostasis.
Autocrine activation by interferon‐γ of STAT factors following T cell activation
Authors: Girdlestone John , Wing Mark
Published: March 2007

The activation of T cells requires engagement of the T cell receptor/CD3 complex and co‐stimulatory molecules, and results in the triggering of several signaling pathways which lead rapidly to the nuclear translocation of several transcription factors, such as nuclear factor (NF)‐ϰB and NF‐AT. A result of this activation process is the induction of a number of genes, including those encoding cytokines such as interleukin‐2, tumor necrosis factor‐α, and interferon (IFN)‐γ which have important immunoregulatory effects. We report here that a DNA‐binding factor containing STAT1 also becomes activated in human peripheral blood T lymphocytes or Jurkat cells, although not until 1–2 h after stimulation. Activation is delayed a further 1–2 h when mononuclear cell cultures are stimulated by an antigen which requires processing. Appearance of the STAT1 factor is significantly reduced in the presence of cyclosporin A, and blocked by cycloheximide, indicating that its activation is dependent upon a protein(s) synthesized in response to initial signaling events. Neutralizing antiserum against IFN‐γ, but not other cytokines tested, blocked activation of the factor almost completely, and IFN‐γ was found in the culture supernatants of stimulated cells at levels at which recombinant IFN‐γ could activate the factor in naive cells. Therefore, a STAT1 transcription factor is activated by IFN‐γ synthesized and released upon stimulation of T lymphocyte populations. While Jurkat cells both secret and respond to IFN‐γ in an autocrine loop, it seems likely that the responding cells may differ from those synthesizing this cytokine in the mononuclear cell cultures in the light of the recent report that Th1 cells lack the IFN‐γ receptor chain necessary for activation of STAT1 (Pernis, A., Gupta, S., Gollob, K. J., Garfein, E., Coffman, R. L., Schindler, C., and Rothman, P., Science 1995. 269: 245).
HIV induces activation of phosphatidylinositol 4‐kinase and mitogen‐activated protein kinase by interacting with T cell CD4 surface molecules
Authors: Schmid‐Antomarchi Heidy , Benkirane Monsef , Breittmayer Violette , Husson Hervé , Ticchioni Michel , Devaux Christian , Rossi Bernard
Published: March 2007

T cell surface CD4 molecules act as co‐receptors that amplify the T cell receptor (TcR)/CD3‐induced signal transduction by a mechanism that requires the interaction of CD4 with p56lck tyrosine kinase (Veillette et al.; Nature 1989. 338: 257). Here, we demonstrate that in the absence of TcR signaling, heat‐inactivated HIV‐1 (HIV‐HI) also elicits a cascade of events generally considered to convey a positive signal, such as protein tyrosine phosphorylation, phosphatidylinositol 4‐kinase and mitogen‐activated protein kinase activation. These results contribute to understand better the control that HIV may exert on its own replication or on T cell apoptosis by modulating the activation status of its target cells through its interaction with T cell surface CD4 molecules.
Cleavage of membrane‐bound CD40 ligand is not required for inducing B cell proliferation and differentiation
Authors: Pietravalle Fabienne , Lecoanet‐Henchoz Sybille , Aubry Jean‐Pierre , Elson Greg , Bonnefoy Jean‐Yves , Gauchat Jean‐Françcois
Published: March 2007

The phycical interaction between the B cell surface molecule CD40 and its ligand, CD40L, is known to be crucial in the development and maintenance of humoral immunity. Recently it has been shown that the CD40L is processed and that its soluble cleavage products are released into the extracellular environment. To study the functions of soluble and membrane‐bound human CD40L on human B cells, we generated an uncleavable CD40L cDNA deletion mutant. The activities of transfectants expressing either mutated or wild‐type CD40L were then compared on human B cells. Both the soluble and the uncleavable membrane‐bound CD40L were able to induce, in conjunction with interleukin‐4, B cell proliferation and IgE synthesis. Therefore, membrane‐bound and soluble CD40L exhibit the same pattern of activities on B cells and membrane CD40L cleavage is not a prerequisite for its function.
CD4 and CD8 expression and T cell antigen receptor gene rearrangement in early intrathymic precursor cells
Authors: Ismaili Jamila , Antica Mariastefania , Wu Li
Published: March 2007

The earliest T precursor population in the adult mouse thymus, considered to have the surface phenotype CD4lo8−3−44+25−Thy‐1lo c‐kit+ (termed the low CD4 precursor), has been shown to have the capacity to produce B cells and dendritic cells, as well as T cells, and to have the T cell antigen receptor (TCR) Cβ gene region in germ‐line configuration. Because of evidence that this precursor population may have low levels of CD8 as well as CD4 on the cell surface, it was isolated, stained for surface CD4 and CD8 and assayed for the expression of messenger RNA (mRNA) for CD4 and CD8 by the reverse transcriptase polymerase chain reaction (RT‐PCR). The low CD4 precursors gave definite, moderate levels of staining for both CD8 and CD4, in contrast to downstream precursors which showed only marginal staining and so could be considered as genuine CD4−8−3− triple negatives. The low CD4 precursor expressed a significant level of mRNA for CD4, indicating that the surface CD4 was produced by these cells. However, the low CD4 precursor did not express a detectable level of mRNA for CD8, suggesting that the surface CD8 was acquired from other cells. Since the low CD4 precursor population was found already to express mRNA for enzymes involved in TCR gene rearrangement, including in this study terminal deoxynucleotidyl transferase (TdT), a PCR procedure was used to assay early precursors for D‐J rearrangements at the TCR β gene locus. However, the low CD4 precursor had the TCR β D‐J genes in germ‐line configuration, D‐J gene rearrangements being first detected several stages downstream in the CD3−4−8−44−25+ precursor population. We conclude that a transient synthesis of CD4, but not of CD8, characterizes these early thymus precursors. Although they have initiated synthesis of some recombination‐associated enzymes, full commitment to the T lineage and TCR gene rearrangement is a later event.
Characterization of mouse CD6 with novel monoclonal antibodies which enhance the allogeneic mixed leukocyte reaction
Authors: Starling Gary C. , Whitney Gena S. , Siadak Anthony W. , Llewellyn Maj‐Britt C. , Bowen Michael A. , Farr Andrew G. , Aruffo Alejandro A.
Published: March 2007

Human CD6 is a cell surface protein expressed by thymocytes, mature T cells, a subset of B cells and certain cells of the brain. On human T cells, CD6 has been shown to act as a co‐stimulatory molecule which modulates T cell receptor (TCR)‐mediated T cell activation. To study further the recently identified mouse CD6 (mCD6), we generated and characterized a set of anti‐mCD6 mAb. Anti‐mCD6 mAb recognizing the mCD6 scavenger receptor cysteine‐rich (SRCR) extracellular domains 1 and 3 were identified. mAb against SRCR domain 3, but not domain 1, inhibited the interaction of CD6 with a recently identified ligand, activated leukocyte cell adhesion molecule (ALCAM). Immunohistochemical analysis indicated that mCD6 expression was largely localized to the T cell areas of lymphoid tissue and, as previously reported in the human, CD6 was also expressed by neurons. CD6 was highly expressed on mouse T cells isolated from the spleen, lymph node and thymus as demonstrated by two‐color immunofluorescence analysis. The CD4+ and CD8+ cells in these lymphoid compartments expressed similar levels of CD6. Immunoprecipitation studies showed that mouse thymocytes predominantly express a CD6 isoform of ∼130 kDa, while splenocytes predominantly express a CD6 isoform of ∼100 kDa. Anti‐mCD6 mAb enhanced allogeneic mixed leukocyte reactions (MLR), indicating that CD6‐ALCAM interactions may regulate the proliferative capacity of T cells.
Time course analysis of α+β+ T cell clones during normal pregnancy
Authors: Höger Torsten A. , Tokuyama Mayumi , Yonamine Kyoko , Hayashi Kazuhiko , Masuko‐Hongo Kayo , Kato Tomohiro , Kobata Tetsuji , Mizushima Yutaka , Nishioka Kusuki , Yamamoto Kazuhiko
Published: March 2007

During normal pregnancy, the fetus continues to mature inside the uterus without rejection. Inherited paternal antigens could be targeted by the maternal immune system. These reactions are believed to play a role in a number of habitual abortions. However, the precise maternal mechanisms preventing fetal tissue rejection are not well understood. Maternal T cells should recognize fetal antigens, so it is conceivable that antigen‐specific T cell response to fetal antigens would occur by proliferation and accumulation of certain T cell clones in the pregnant mother. To elucidate the maternal immune response to the fetus, we investigated the clonality of expanded T cells in peripheral blood lymphocytes in ten normal pregnant women. We employed reverse transcriptase‐polymerase chain reaction for T cell receptor β chain gene and subsequently analyzed the PCR product by single‐strand conformation polymorphism analysis. A large number of distinctly expanded T cell clones were detected during pregnancy. These accumulations were observed as early as the ninth to tenth week post‐conception and reached a maximum during the second trimester, suggesting the existence of dynamic antigen‐specific T cell responses in the pregnant mother. However, after the 30th week of gestation, nearly all expanded T cell clones disappeared before parturition and the degree of clonality reached almost normal levels. Our results clearly indicate the existence of dynamic maternal T cell responses during pregnancy.
Increase of intracellular calcium is the essential signal for the expression of CD40 ligand
Authors: Nüsslein Hubert G. , Frosch Karl‐Heinz , Woith Walter , Lane Peter , Kalden Joachim R. , Manger Bernhard
Published: March 2007

CD40 ligand (CD40L) is present on activated but not on resting T cells. In contrast to the activation markers CD25 and CD71, a strong CD40L expression could be induced by calcium ionophore alone but not by phorbol 12‐myristate 13‐acetate (PMA). Ionomycin induced a very early mRNA and protein surface expression of CD40L within the first 2 h, whereas CD25 and CD71 did not appear earlier than 6 h after stimulation. The mitogens phytohemagglutinin and concanavalin A induced little CD40L, but together with PMA, a markedly increased CD40L expression was observed. In T cells stimulated with immobilized anti‐CD3, co‐stimulation with anti‐CD28 or PMA induced an earlier and higher maximal CD40L expression. CD40L expression of purified T cells was higher and more prolonged compared to that of T cells in unseparated peripheral blood mononuclear cells. We conclude that the expression of CD40L on T cells is profoundly different from other early activation markers with regard to signal requirements, kinetics and the role of accessory cells in the system.
Distribution of α4β7 and αEβ7 integrins on thymocytes, intestinal epithelial lymphocytes and peripheral lymphocytes
Authors: Andrew David P. , Rott Lusijah S. , Kilshaw Peter J. , Butcher Eugene C.
Published: March 2007

β7 is expressed on subsets of thymocytes, while T and B lymphocytes show heterogeneous expression of β7. Here, we examine the phenotype of the thymocyte and lymphocyte subsets which express α4β7 and αEβ7 using mAb against αEβ7 and mAb DATK32 which recognizes a combinatorial epitope on α4β7. β7+ thymocytes have a mature phenotype: TcR+, CD11ahi CD44hi HSAdull. Small subsets of double‐negative CD4−CD8−, single‐positive CD4+ and CD8+ thymocytes express α7, while double‐positive CD4+ CD8+ thymocytes are β7. However, two integrins αEβ7 and α4β7 recognized by anti‐β7 are not expressed on an identical subpopulation of thymocytes, as βEα7+α4β7−, αEβ7+α4β7+ and αEβ7−α4β7+ thymocyte subsets are evident. Similarly, intraepithelial lymphocytes express high levels of αEβ7 but little α4β7. In the spleen, Peyer's patches and lymph nodes, α4β7 is expressed at higher levels on most B lymphocytes than on the majority of T lymphocytes, while a small subset of T lymphocytes, which includes both CD4+ and CD8+ lymphocytes, express high levels of β7 in the form of α4β7 and αEβ7, although, as observed with lymphocytes, not all α4β7hi CD4− lymphocytes expressed α4β7. The population of α4β7hi CD4 lymphocytes are enriched in Peyer's patches and form subsets of the memory CD4+ lymphocyte population, which can be further subdivided on the basis of αEβ7, L‐selectin and α4 expression. Therefore, memory CD4+ lymphocytes are highly heterogeneous in their expression of adhesion receptors, and presumably these subpopulations will exhibit very different trafficking properties.
The expansion and selection of T cell receptor αβ intestinal intraepithelial T cell clones
Authors: Regnault Armelle , Levraud Jean‐Pierre , Lim Annick , Six Adrien , Moreau Christiane , Cumano Ana , Kourilsky Philippe
Published: March 2007

In conventional mice, the T cell receptor (TCR)αβ+ CD8αα+ and CD8αβ+ subsets of the intestinal intraepithelial lymphocytes (IEL) constitute two subpopulations. Each comprise a few hundred clones expressing apparently random receptor repertoires which are different in individual genetically identical mice (Regnault, A., Cumano, A., Vassalli, P., Guy‐Grand, D. and Kourilsky, P., J. Exp. Med. 1994. 180: 1345).

We analyzed the repertoire diversity of sorted CD8αα and CD8αβ+ IEL populations from the small intestine of individual germ‐free mice that contain ten times less TCRαβ+ T cells than conventional mice. The TCRβ repertoire of the CD8αα and the CD8αβ IEL populations of germ‐free adult mice shows the same degree of oligoclonality as that of conventional mice. These results show that the intestinal microflora is not responsible for the repertoire oligoclonality of TCRαβ+ IEL. The presence of the microflora leads to an expansion of clones which arise independently of bacteria. To evaluate the degree of expansion of IEL clones in conventional mice, we went on to measure their clone sizes in vivo by quantitative PCR in the total and in adjacent sections of the small intestine of adult animals. We found that both the CD8αα and the CD8αβ TCRαβ IEL clones have a heterogeneous size pattern, with clones containing from 3 × 103 cells up to 1.2 × 106 cells, the clones being qualitatively and quantitatively different in individual mice. Cells from a given IEL clone are not evenly distributed throughout the length of the small intestine. The observation that the TCRαβ IEL populations comprise a few hundred clones of very heterogeneous size and distribution suggests that they arise from a limited number of precursors, which may be slowly but continuously renewed, and undergo extensive clonal expansion in the epithelium.
Differential regulation of human T cell responsiveness by mucosal versus blood monocytes
Authors: Qiao Liang , Braunstein Jutta , Golling Markus , Schürmann Guido , Autschbach Frank , Möller Peter , Meuer Stefan
Published: March 2007

Human intestinal T lymphocytes are constantly exposed to a large number of foreign antigens without developing a systemic immune response. One crucial mechanism leading to this intestinal hyporesponsiveness is based on impaired signal transduction through the T cell receptor/CD3 complex in lamina propria T lymphocytes (LP‐T). In this study, we addressed the question whether a lack of co‐stimulatory/progression signals might also contribute to LP‐T hyporesponsiveness. To this end, isolated human monocyte populations from the intestinal lamina propria were obtained and their phenotypes as well as their capacity to promote T cell activation studied. Here, we demonstrate that lamina propria macrophages (LP‐MO), in contrast to peripheral blood monocytes (PB‐MO), do not support proliferation of either LP‐T or PB‐T. This may be due to the low expression of ligands (CD54, CD58, CD80) for the T cell accessory receptors CD11/18, CD2 and CD28/CTLA‐4 on mucosal macrophages. Thus, down‐regulation of both recognition/competence and co‐stimulatory/progression signals contribute to intestinal hypo‐ or unresponsiveness.
Specificity of an HLA‐DRB1*0401‐restricted T cell response to type II collagen
Authors: Fugger Lars , Rothbard Jonathan B. , Sonderstrup‐McDevitt Grete
Published: March 2007

A panel of HLA‐DRB1*0401‐restricted CD4+ mouse T cell hybridomas specific for bovine type II collagen were generated from transgenic mice expressing the human HLA‐DRA1*0101/‐DRB1*0401 and CD4 molecules. The vast majority recognized a single peptide determinant corresponding to residues 261–273 (CII 261–273). This determinant was rapidly defined by the use of a predictive algorithm for peptide binding to DRB1*0401. CII 261–273 is conserved in bovine and human type II collagen and overlaps with an important I‐Aq‐restricted T cell determinant in mice with collagen‐induced arthritis. This study demonstrates how HLA‐DR and human CD4‐transgenic mice can be used to identify a T cell epitope in a potential or candidate autoantigen.
Proliferative recruitment of intestinal intraepithelial lymphocytes after microbial colonization of germ‐free mice
Authors: Imaoka Akemi , Matsumoto Satoshi , Setoyama Hiromi , Okada Yasushi , Umesaki Yoshinori
Published: March 2007

Intraepithelial lymhocytes (IEL), particularly αβ TCR‐bearing IEL (αβ‐IEL), dramatically increase in number after microbial colonization of formerly germ‐free (ex‐GF) mice (Umesaki et al., Immunology 1993. 79: 32). In this study, the kinetics of expansion of IEL after microbial colonization in ex‐GF mice were investigated by the bromodeoxyuridine (BrdUrd) continuous labeling method. In GF mice, γδ‐ and αβ‐IEL were gradually labeled with BrdUrd, reaching approximately 30% and 15% labeling, respectively, after 10 days of continuous BrdUrd labeling. In conventional (CV) mice, the percentage of BrdUrd‐labeled αβ‐IEL was a little higher than that for γδ‐IEL. The maximal labeling for αβ‐IEL and γδ‐IEL reached 50–60% and 40%, respectively, in 10 days. In the case of conventionalized ex‐GF mice, continuous labeling was started 11 days after microbial colonization of GF mice because αβ‐IEL outnumbered γδ‐IEL during this period. In this case, 75% of αβ‐IEL and 67% of γδ‐IEL were labeled with BrdUrd in 10 days. On the other hand, the apparent half lives of αβ‐IEL and γδ‐IEL were 10 and 20 days, respectively, in CV mice. These results strongly suggest that the number of IEL, particularly αβ‐IEL, increases after microbial colonization through recruitment into the cell cycle of a large proportion of IEL or their immediate precursors. The difference in the relative BrdUrd‐labeling rate of αβ‐IEL to that of γδ‐IEL between germ‐free and conventionalized mice indicates a preferential increase in the αβ‐IEL subset during the course of conventionalization.
Recognition of altered self major histocompatibility complex molecules modulated by specific peptide interactions
Authors: Nepom Gerald T. , Ou Dawei , Lybrand Terry P. , Deweese Carol , Domeier Mary Ellen , Buckner Jane H. , Mitchell Leslie A. , Tingle Aubrey J.
Published: March 2007

Antigen‐specific and major histocompatibility complex (MHC)‐restricted recognition by the T cell receptor involves multiple structural contacts over a large molecular surface area. Using a human T cell clone specific for a rubella viral peptide restricted by subsets of HLA DR4 molecules, we identified structurally diverse combinations of peptide‐MHC complexes which were functionally equivalent for T cell recognition. Presentation of the rubella‐derived peptide on DR4 molecules with an E‐74 polymorphism triggered T cell recognition, as did presentation of a single amino acid‐substituted peptide in the context of the DR4 molecule which lacked the E‐74 site. Peptide binding and molecular modeling analysis indicates the structural and functional complementarity of T cell recognition for a specific amino acid side chain, whether contributed by the peptide or by the MHC molecule.
T lymphocytes from the normal human peritoneum contain high frequencies of Th2‐type CD8+ T cells
Authors: Birkhofer Andreas , Rehbock Joachim , Fricke Harald
Published: March 2007

The majority of peritoneal T lymphocytes have been shown to be CD8+ and to co‐express CDw60. Expression of CDw60 characterizes CD8 T cells capable of secreting interleukin (IL)‐4 and supporting IgG production by B cells. We analyzed at the clonal level the functional cytokine profile of CD8+ T lymphocytes from the normal human peritoneum. While the majority of the clones produced interferon (IFN)‐γ and exhibited high alloantigen‐specific cytolytic activity, some clones secreted IL‐4 and IL‐5 but no detectable IFN‐γ. These Th2‐type CD8+ T cell clones provided substantial B cell help for IgG and IgA synthesis and exhibited reduced cytolytic activity. Our results suggest that distinct subsets of CD8+ T cell may occur in different immune compartments.
A promiscuous T cell hybridoma restricted to various I‐A molecules
Authors: Katoh Masahito , Itoh Yasushi , Ogasawara Kazumasa , Kajino Kiichi , Nishihori Hiroki , Takahashi Akio , Matsuki Naoto , Iwabuchi Kazuya , Yoshida Takato O. , Good Robert A. , Onoé Kazunori
Published: March 2007

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43‐58 peptide. Positions 46 and 54 of p43‐58 were shown to be the MHC‐binding sites. Specific amino acids were identified on the MHC‐binding sites which bound to the relevant I‐A molecule. In the present study, using NOD (I‐Ag7) mice, we established a T cell hybridoma, NOE33‐1‐2, specific for a p43‐58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33‐1‐2 recognized 46R50E54A in the presence of not only I‐Ag7, but also I‐Ad, s, u and v. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA‐DR or I‐E molecules consist of monomorphic α and polymorphic β chains, the promiscuous T cell clone NOE33‐1‐2 recognized peptides with various I‐A molecules lacking the monomorphic chain.
The myelin basic protein‐specific T cell repertoire in (transgenic) Lewis rat/SCID mouse chimeras: preferential Vβ8.2 T cell receptor usage depends on an intact Lewis thymic microenvironment
Authors: Kääb Georg , Brandl Gerhard , Marx Alexander , Wekerle Hartmut , Bradl Monika
Published: March 2007

In the Lewis rat, myelin basic protein (MBP)‐specific, encephalitogenic T cells preferentially recognize sequence 68–88, and use the Vβ8.2 gene to encode their T cell receptors. To analyze the structural prerequisites for the development of the MBP‐specific T cell repertoire, we reconstituted severe‐combined immunodeficient (SCID) mice with fetal (embryonic day 15–16) Lewis rat lymphoid tissue, and then isolated MBP‐specific T cell lines from the adult chimeras after immunization. Two types of chimera were constructed: SCID mice reconstituted with rat fetal liver cells only, allowing T cell maturation within a chimeric SCID thymus consisting of mouse thymic epithelium and rat interdigitating dendritic cells, and SCID mice reconstituted with rat fetal liver cells and rat fetal thymus grafts, allowing T cell maturation within the chimeric SCID and the intact Lewis rat thymic microenvironment. Without exception, the T cell lines isolated from MBP‐immunized SCID chimeras were restricted by MHC class II of the Lewis rat (RT1.B1), and none by I‐Ad of the SCID mouse. Most of the T cell lines recognized the immunodominant MBP epitope 68–88. In striking contrast to intact Lewis rats, in SCID mice reconstituted by rat fetal liver only, MBP‐specific T cell clones used a seemingly random repertoire of Vβ genes without a bias for Vβ8.2. In chimeras containing fetal Lewis liver plus fetal thymus grafted under the kidney capsule, however, dominant utilization of Vβ8.2 was restored. The migration of liver‐derived stem cells through rat thymus grafts was documented by combining fetal tissues from wild‐type and transgenic Lewis rats. The results confirm that the recognition of the immunodominant epitope 68–88 by MBP‐specific encephalitogenic T cells is a genetically determined feature of the Lewis rat T cell repertoire. They further suggest that the formation of the repertoire requires T cell differentiation in a syngeneic thymic microenvironment.
Identification of a novel subpopulation of germinal center B cells characterized by expression of IgD and CD70
Authors: Lens Susanne M. A. , Keehnen Robert M. J. , van Oers Marinus H. J. , van Lier René A. W. , Pals Steven T. , Koopman Gerrit
Published: March 2007

CD27, which belongs to the tumor necrosis‐factor receptor family, is expressed on germinal center (GC) but not on naive B cells, suggesting an important function of this molecule in the regulation of the GC reaction. We described here the expression of CD70, which is the ligand for CD27. We observed that in most tonsils, CD70 is only expressed on part of the IgD−, CD38− B cell population, which have been described as memory B cells. However, in 10 % of the tonsils tested, CD70+ IgD+ GC were found. The CD70+ GC B cells were small cells that also expressed CD44 and CD39, but were CD10− and CD38−, suggesting that they represent very recent immigrants that are in the process of forming a GC. The concordant expression of CD27 and its ligand CD70 on this primordial subset of GC B cells suggests an important role for CD27/CD70 interaction at this stage of GC formation.
CD3‐induced apoptosis of CD4+CD8+ thymocytes in the absence of clonotypic T cell antigen receptor
Authors: Tokoro Yayoi , Tsuda Shoko , Tanaka Shigeko , Nakauchi Hiromitsu , Takahama Yousuke
Published: March 2007

Clonal selection of T cells mediated through the T cell antigen receptor (TCR) mostly occurs at the CD4+CD8+ double positive thymocyte stage. Immature CD4+CD8+ thymocytes expressing self‐reactive TCR are induced to die upon clonotypic engagement of TCR by self antigens. CD3 engagement by antibody of the surface TCR‐CD3 complex is known to induce apoptosis of CD4+CD8+ thymocytes, a process that is generally thought to represent antigen‐induced negative selection in the thymus. The present study shows that the CD3‐induced apoptosis of CD4+CD8+ thymocytes can occur even in TCRα− mutant mice which do not express the TCRαβ/CD3 antigen receptor. Anti‐CD3 antibody induces death of CD4+CD8+ thymocytes in TCRα− mice either in cell cultures or upon administration in vivo. Interestingly, most surface CD3 chains expressed on CD4+CD8+ thymocytes from TCRα− mice are not associated with clonotypic TCR chains, including TCRβ. Thus, apoptosis of CD4+CD8+ thymocytes appear to be induced through the CD3 complex even in the absence of clonotypic antigen receptor chains. These results shed light on previously unknown functions of the clonotype‐independent CD3 complex expressed on CD4+CD8+ thymocytes, and suggest its function as an apoptotic receptor inducing elimination of developing thymocytes.
Ribozyme modulation of lipopolysaccharide‐induced tumor necrosis factor‐α production by peritoneal cells in vitro and in vivo
Authors: Sioud Mouldy
Published: March 2007

We have utilized synthetic ribozymes to modulate the lipopolysaccharide (LPS)‐induced production of tumor necrosis factor‐α (TNF‐α) by peritoneal cells. Two hammerhead ribozymes (mRz1 and mRz2) were prepared by transcription in vitro and their activities in vitro and in vivo were investigated. Both ribozymes cleaved their RNA target with an apparent turnover number (kcat) of 2 min−1, and inhibited TNF‐α gene expression in vitro by 50% and 70%, respectively. When mRz1 and mRz2, entrapped in liposomes, were delivered into mice by intraperitoneal injection, they inhibited LPS‐induced TNF‐α gene expression in vivo with mRz2 being the most effective. This enhanced activity could result from the facilitation of catalysis by cellular endogenous proteins, since they specifically bind to mRz2 as compared to mRz1. Furthermore, a significant mRz2 activity can be recovered from peritoneal cells 2 days post‐administration in vivo. The anti‐TNF‐α ribozyme treatment in vivo resulted in a more significant reduction of LPS‐induced IFN‐γ protein secretion compared to IL‐10. In contrast to this pleiotropic effect, the anti‐TNF‐α ribozyme treatment did not affect the heterogenous expression of Fas ligand by peritoneal cells, indicating the specificity of the treatment. Taken together, the present data indicate that the biological effects of TNF‐α can be modulated by ribozymes. In addition, the data suggest that ribozymes can be administered in a drug‐like manner, and therefore indicate their potential in clinical applications.
Peptides derived from IgE heavy chain constant region induce profound IgE isotype‐specific tolerance
Authors: Wang Yanyu , Schmaltz Robert , Liu Fu‐Tong , Robertson Michael W. , Petro Thomas M. , Chen Swey‐Shen
Published: March 2007

(BALB/c × SJL)F1 mice, perinatally injected with peptide‐N‐glyconase F‐treated, deglycosylated IgE heavy chain or recombinant IgE heavy chain (CHϵ2‐CHϵ4), were profoundly inhibited in antigen‐specific IgE production. There exist minimally two tolerogenic IgE peptides, residing in the CHϵ2 and CHϵ4 domains. Peptide I, generated by V8 protease, comprises 39 amino acids within CHϵ2, beginning at amino acid 103. Peptide E begins at amino acid 312 of the CHϵ4 domain and extends through the CHϵ4 domain. The total lack of antigen‐specific IgE responses in IgE peptide‐treated mice was not due to overproduction of interferon‐γ, nor lack of interleukin (IL)‐4, as predicted by the Th2/IL‐4 paradigm for IgE production. IgE‐tolerant mice exhibited comparable levels of circulating anti‐IgE antibodies to those of PBS‐treated control mice. IgG obtained from sera of both sources failed to inhibit IgE responses in vitro. Moreover, IgE responses of spleen cells from IgE peptides‐treated mice were restored by CD4+ T cells from PBS‐treated control mice. We hypothesize that regulation of antigen‐specific IgE responses is mediated by CD4+ T cells which normally recognize IgE peptides on IgE precursor B cells, and can be rendered tolerant by perinatal IgE peptide treatment.
Development in vitro of human CD4+ thymocytes into functionally mature Th2 cells. Exogenous interleukin‐12 is required for priming thymocytes to produce both Th1 cytokines and interleukin‐10
Authors: Mingari Maria Cristina , Maggi Enrico , Cambiaggi Anna , Annunziato Francesco , Schiavetti Francesca , Manetti Roberto , Moretta Lorenzo , Romagnani Sergio
Published: March 2007

Fresh postnatal thymocyte cell suspensions were directly cloned under limiting dilution conditions with either phytohemagglutinin or toxic shock syndrome toxin‐1 (TSST‐1), a bacterial superantigen. Cultures contained allogenic irradiated feeder cells and interleukin (IL)‐2, in the absence or presence of exogenous IL‐4, interferon (IFN)‐γ or IL‐12. The resulting CD4+ T cell clones generated under these different experimental conditions were then analyzed for their ability to produce IL‐2, IL‐4, IL‐5, IL‐10, IFN‐γ and tumor necrosis factor (TNF)‐β in response to stimulation with phorbol 12‐myristate 13‐acetate (PMA)+anti‐CD3 monoclonal antibody or PMA + ionomycin. Different from T cell clones generated from peripheral blood, virtually all CD4+ T cell clones generated from human thymocytes produced high concentrations of IL‐2, IL‐4 and IL‐5, but no IFN‐γ, TNF‐β or IL‐10. Moreover, after activation, these clones expressed on their surface membrane both CD30 and CD40 ligand, but not the product of lymphocyte activation gene (LAG)‐3, and provided strong helper activity for IgE synthesis by allogeneic B cells. The Th2 cytokine pattern could not be modified by the addition of IFN‐γ. However, upon addition of exogenous IL‐12, the resulting CD4+ thymocyte clones produced TNF‐β, IFN‐γ, and IL‐10 in addition to IL‐4 and IL‐5. These results suggest that CD4+ human thymocytes have the potential to develop into cells producing the Th2 cytokines IL‐4 and IL‐5, whereas the ability to produce both Th1 cytokines and IL‐10 is acquired only after priming with IL‐12.
T cell tolerance and activation to a transgene‐encoded tumor antigen
Authors: Antoniou Antony , McCormick David , Scott Diane , Yeoman Helen , Chandler Phillip , Mellor Andrew , Dyson Julian
Published: March 2007

Much has been learned in recent years concerning the nature of tumor antigens recognized by T cells. To apply this knowledge clinically, the nature of the host response to individual and multiple tumor antigens has to be characterized. This will help to define the efficacy of immune surveillance and the immune status of the host following exposure to tumor antigens expressed on pre‐neoplastic tissue. To approach these questions, we have developed a transgenic mouse which expresses the tumor‐specific antigen P91A. The single amino acid substitution in P91A results in the expression of a new MHC class I (H‐2Ld)‐binding peptide. In transgenic tissue, the H‐2Ld/P91A complex is expressed in isolation from other tumor‐associated antigens, allowing definition of the immune response to a single defined tumor antigen, a situation closely analogous to events during tumorigenesis. We show that CD8+ T cell immune surveillance of P91A is ineffective without the introduction of a helper determinant operating through stimulation of CD4+ T cells. Recognition of the isolated P91A tumor antigen on normal tissue by CD8+ T cells is a tolerogenic process. Induction of T cell tolerance suggests tumor antigen‐T cell interactions occurring during tumorigenesis may elicit T cell tolerance and hence confound some immunotherapeutic approaches.
Lack of detectable defect in DNA double‐strand break repair and DNA‐dependent protein kinase activity in radiosensitive human severe combined immunodeficiency fibroblasts
Authors: Nicolas Nathalie , Finnie Nicholas J. , Cavazzana‐Calvo Marina , Papadopoulo Dora , Le Deist Françoise , Fischer Alain , Jackson Stephen P. , de Villartay Jean‐Pierre
Published: March 2007

The initial step of the V(D)J recombination occurs through the generation of a DNA double‐strand break (dsb). Defects in the DNA‐dependent protein kinase complex (DNA‐PK) result in an inability to perform either V(D)J recombination or any dsb repair effectively. The human autosomal T‐B‐severe combined immunodeficiency (SCID) condition is characterized by an absence of both B and T lymphocytes and is accompanied in some patients by an increase in γ‐ray sensitivity (T‐B‐RS SCID) comparable to that found in mouse SCID cells. We show here that cells from six patients with T‐B‐RS SCID had normal DNA‐dsb repair kinetics. Furthermore, DNA‐PK activity was present in extracts from these human T‐B‐RS SCID fibroblasts. We therefore conclude that some human T‐B‐RS SCID disorders are not caused by a defect in an essential DNA‐PK component.
Recognition of out‐of‐frame major histocompatibility complex class I‐restricted epitopes in vivo
Authors: Elliott Tim , Bodmer Helen , Townsend Alain
Published: March 2007

In the course of constructing a recombinant vaccinia virus encoding the influenza A nucleoprotein (NP) gene preceeded by the hemagglutinin leader sequence, we isolated a single base‐pair deletion mutant which gave rise to L+NP(1–159) in which only the first 159 amino acids were in frame. Despite this, when we infected target cells, we found that the point mutant was able to sensitize them for lysis not only by cytotoxic T cells recognizing residues 50–58 (the in‐frame portion), but also by CTL to epitopes which are downstream of the mutation (366–374 and 378–386). Furthermore, normal C57BL/6 mice can be primed with the frameshift NP to recognize the immunodominant Db‐restricted epitope 366–374 (which is out of frame). Experiments in which the mutant gene product was processed in the endoplasmic reticulum of target cells suggested that the apparent suppression occured during polypeptide extension.
T cell major histocompatibility complex class II molecules down‐regulate CD4+ T cell clone responses following LAG‐3 binding
Authors: Huard Bertrand , Prigent Philippe , Pagès Franck , Bruniquel Denis , Triebel Frédéric
Published: March 2007

T cell response to its antigen requires recognition by the T cell receptor together with a co‐receptor molecule, either CD4 or CD8. Additional molecules have been identified that are capable of delivering the co‐stimulatory signals provided by APC. Following T cell priming, a number of T cell activation antigens are expressed that may play a role in the inactivation phase of the T cell response. The lymphocyte activation gene (LAG)‐3 protein and its counter‐receptors, the major histocompatibility complex (MHC) class II molecules, are such activation antigens whose interaction may result in the down‐regulation of the ongoing immune response. To investigate the role of LAG‐3/class II molecule interaction, we produced a soluble form of LAG‐3 by fusing the extracellular Ig domains of this membrane protein to the constant region of human IgG1 (LAG‐31g). Here, we show a direct and specific binding of LAG‐3Ig to class II molecules on the cell surface. In addition, we show that LAG‐3/class II molecule interaction leads to the down‐regulation of CD4+ Ag‐specific T cell clone proliferation and cytokine secretion. This inhibitory effect is observed at the level of the effector cells and not the APC and is also found with anti‐CD3 mAb, PHA + PMA or low‐dose IL‐2 driven stimulation in the absence of APC. These functional studies indicate that T cell MHC class II molecules down‐regulate T cell proliferation following LAG‐3 binding and suggest a role for LAG‐3 in the control of the CD4+ T cell response.
Interleukin‐12 induces interferon‐γ‐dependent switching of IgG alloantibody subclass
Authors: Gracie J. Alastair , Bradley J. Andrew
Published: March 2007

Interleukin‐12 (IL‐12) is a key immunoregulatory cytokine which promotes cell‐mediated immunity. Its influence on the humoral immune response is less clearly defined. In this study, the effect of systemic IL‐12 treatment on the T cell‐dependent humoral immune response in the rat was examined using an experimental system in which PVG‐RT1u congenic rats were immunized with class I Aa alloantigen in the form of a blood transfusion from the recombinant PVG.R8 rat strain. Administration of IL‐12 following allo‐immunization augmented the humoral immune response as determined by increased levels of cytotoxic anti‐class I major histocompatibility complex antibodies. However, the effect of IL‐12 on individual IgG isotypes was highly selective. Levels of allospecific antibodies of the IgG2b and IgG2c subclasses were markedly increased, whereas IgG1 alloantibody levels were profoundly reduced. The observed alterations in alloantibody response were dependent, in large part, on the stimulatory effect of IL‐12 on interferon (IFN)‐γ production by T lymphocytes and natural killer cells, since they were abrogated by co‐administration of neutralizing anti‐IFN‐γ monoclonal antibody following alloantigen immunization.
Involvement of p72syk kinase, p53/561yn kinase and phosphatidyl inositol‐3 kinase in signal transduction via the human B lymphocyte antigen CD22
Authors: Tuscano Joseph M. , Engel Pablo , Tedder Thomas F. , Agarwal Alka , Kehrl John H.
Published: March 2007

CD22 is a B lymphocyte‐specific membrane protein that functions as an adhesion molecule via its interactions with a subset of α2‐6‐linked sialic acid‐containing glycoproteins. Engagement of CD22 with a monoclonal antibody (HB22.23) that blocks the binding of CD22 to its ligands results in rapid CD22 tyrosine phosphorylation and in increased association of CD22 with p53/561yn kinase, p85 phosphatidyl inositol‐3 kinase, and p72syk kinase. Synthetic peptides that span various regions of the intracellular portion of CD22 were used to map potential kinase binding sites. All three kinases associated with a tyrosine‐phosphorylated peptide that spans tyrosine amino acid residues 822 and 842, implicating this as an important region in mediating CD22 signal transduction. In addition, purified p56lyn directly bound to the same peptide. Engagement of CD22 with HB22.23 was sufficient to stimulate normal B cell proliferation. This study further substantiates the importance of CD22 as a B lymphocyte signaling molecule and begins to unravel the mechanisms by which CD22 cross‐linking can alter B cell function.
Selective inhibition of interleukin‐1β gene expression in activated RAW 264.7 macrophages by interferon‐γ
Authors: Chujor Chujor S. N. , Klein Lilian , Lam Charles
Published: March 2007

The ability of interleukin‐1 (IL‐1) to activate epidermal cell populations supports its role as a key cytokine in the pathogenesis of a number of inflammatory skin diseases. In the present study, we have examined the effect of interferon (IFN)‐γ on the expression of the IL‐β gene in mouse RAW 264.7 macrophages activated by lipopolysaccharide (LPS) plus tumor necrosis factor (TNF)‐α. Incubation of macrophages with both LPS and TNF‐α resulted in the expression of both IL‐1β and inducible nitric oxide synthase (iNOS) mRNA transcripts and increased the release of IL‐1β protein and nitrite production in culture supernatants. Addition of IFN‐γ up‐regulated the expression of the iNOS gene in cells activated by LPS + TNF‐α, but significantly suppressed the induction of IL‐1β gene expression in a dose‐dependent manner. The suppression required neither de novo protein synthesis nor involved destabilization of the mRNA transcripts. Together, these findings suggest that IFN‐γ can be an important regulatory cytokine in a chronic inflammatory site and may explain its purported anti‐inflammatory effects in certain dermatological diseases.