bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of entire myelin basic protein‐induced experimental autoimmune encephalomyelitis in Lewis rats by major histocompatibility complex class II‐binding competitor peptides
Authors: Wauben Marca H. M. , Kozhich Alexander , Joosten Irma , Schlief Angelique , Boog Claire J. P. , Van Eden Willem
Published: March 2007

Previous studies have shown that major histocompatibility complex (MHC) blockade by competitor peptides with high MHC class II binding affinity can prevent peptide‐induced experimental autoimmune encephalomyelitis (EAE). However, none of these studies addressed the question whether this approach could also be used to prevent EAE induced with a multivalent antigen. In this report we show the effect of competitor peptides co‐immunized during EAE induction with entire guinea pig myelin basic protein (MBP) in Lewis rats. As MHC class II binding competitor peptides we used one nonimmunogenic disease‐nonrelated peptide, and two immunogenic peptides, one EAE‐related and one non‐EAE‐related. The respective efficacy of these three competitor peptides to inhibit MBP‐induced proliferation of an encephalitogenic T cell line in vitro correlated with their respective MHC binding affinity. Co‐immunization of the competitor peptides during disease induction with entire MBP resulted in a competitor concentration‐dependent inhibition of clinical signs of EAE. These results demonstrate that, although polyclonal T cell responses to MBP were not completely inhibited, co‐administration of immunogenic or nonimmunogenic either EAE‐related or non‐EAE‐related MHC class II binding competitor peptides can inhibit the development of EAE induced with entire MBP.
CD3−CD8+ intestinal intraepithelial lymphocytes (IEL) and the extrathymic development of IEL
Authors: Lin Tesu , Matsuzaki Goro , Yoshida Hiroki , Kobayashi Noritada , Kenai Hiroyuki , Omoto Kazuya , Nomoto Kikuo
Published: March 2007

Present evidence suggests that a majority of murine CD3+ intraepithelial intestinal lymphocytes (IEL) are extrathymically derived T cells and that these extrathymically derived IEL phenotypically express the CD8 homodimer (CD8αα). Recently, CD3− IEL have been reported to express the recombination activating gene (RAG‐1), suggesting that precursors to extrathymically derived CD3+CD8+αα IEL exist on the intestinal epithelium. To study in detail whether these CD3− IEL can develop into CD3+CD8+αα IEL, we analyzed the CD3− IEL subset and found that it can be separated into two subsets, namely CD3−CD8− and CD3−CD8+ IEL. We show that (1) CD3−CD8− IEL are mostly small, non‐granular and phenotypically Pgp‐1+ IL‐2R+ B220−, while CD3−CD8+ IEL are mostly large, granular and phenotypically Pgp‐1− IL‐2R+ B220+, (2) CD3−‐CD8+ IEL express the RAG‐1 gene, and (3) CD3−CD8−, CD3−CD8+ and CD3+CD8+αα IEL, respectively, appear sequentially in normal ontogeny and in bone marrow‐reconstituted thymectomized radiation chimeras. In the latter, virtually all CD3+CD8+αα IEL expressed the γδ T cell receptor (TCR), but not the αβ TCR. From this and what is presently known about T cell development, we propose that CD3−CD8+ IEL are an intermediate in extrathymic IEL development and that the development of extrathymically derived IEL occurs at the intestinal epithelium from CD3−CD8− to CD3−CD8+ to CD3+(γδ TCR)CD8+αα.
Flow cytometric determination of cytokines in activated murine T helper lymphocytes: Expression of interleukin‐10 in interferon‐γ and in interleukin‐4‐expressing cells
Authors: Assenmacher Mario , Schmitz Jürgen , Radbruch Andreas
Published: March 2007

In an immune response, effector functions are controlled by T helper (Th) 1 cytokines [interferon‐γ (IFN‐γ), interleukin (IL)‐2 and tumor necrosis factor‐β] and Th 2 cytokines (IL‐4, IL‐5 and IL‐10). Here we analyze by multiparameter immunofluorescence to what extent IL‐2, IL‐4, IL‐5, IL‐10 and IFN‐γ are co‐expressed in individual normal murine Th cells upon activation in vitro with the bacterial superantigen Staphylococcus aureus enterotoxin B, presented in the context of major histocompatibility complex class II. IL‐2 and IFN‐γ are co‐expressed by some, but not by other Th cells. Expression of IL‐4 and IFN‐γ is exclusive. IL‐10 is co‐expressed in individual cells either with IL‐4 or with IFN‐γ. No IL‐5‐expressing cells are detected. While IL‐10‐ and IL‐4‐co‐expressing Th cells correspond to classical Th 2 cells, cells co‐expressing IL‐10 and IFN‐γ could be involved in negative‐feedback regulation of a Th 1 response. Apart from such functional implications, our results show that IL‐2, IL‐4, IL‐5, IL‐10 and IFN‐γ are expressed independently of each other in individual murine Th cells.
Maternal transfer of infectious mouse mammary tumor retroviruses does not depend on clonal deletion of superantigen‐reactive Vβ14+ T cells
Authors: Penninger Josef M. , Wallace Valerie A. , Timms Emma , Mak Tak W.
Published: March 2007

Female C3H/HeJ mice maternally transmit through their milk an infectious mouse mammary tumor retrovirus (MMTV) which causes clonal deletion of T cell receptor (TcR)Vβ14+ T cells reactive to the retroviral superantigen (SAG). To test whether CD4+ or CD8+ T cells are crucial for intestinal infection and maternal transfer of exogenous retroviruses, newborn mice lacking CD4 or CD8 molecules after gene targetting were raised by surrogate C3H/HeJ mothers. In CD8−/− mice, clonal deletion of TcRVβ14+ cells reactive to the SAG from this exogenous MMTV occured with delayed kinetics. Deletion of TcRVβ14+ cells was not observed in CD4−/− mice up to 12 months after exposure to the retrovirus. In both CD4−/− and CD8−/− mice TcRVβ5+ and TcRVβ11+ T cells were deleted in the presence of genomically integrated endogenous MMTV (Mtv), indicating that the lack of SAG‐induced clonal deletion was not due to a general defect in these mutant mouse strains. Although TcRVβ14+ T cells were not deleted in CD4−/− mice, female CD4−/− mice nursed on C3H/HeJ milk maternally transmitted the retrovirus to their offspring, albeit with delayed kinetics. These data demonstrate that CD4+ and CD8+ lymphocytes influence clonal deletion events and that the mechanisms responsible for clonal deletion of SAG‐reactive TcRVβ14+ T cells may be different from mechanisms which allow the mammary tumor virus to enter the mammary gland and complete its infectious cycle.
Thymocyte apoptosis induced by elevated endogenous corticosterone levels
Authors: Gruber Johann , Sgonc Roswitha , Hu Yan Hua , Beug Hartmut , Wick Georg
Published: March 2007

A well‐known model of apoptosis is induction in thymocytes by injection of pharmacological doses of exogenous steroids. The aim of this study was to investigate whether this process also occurs under physiological conditions, i.e. by stimulation of endogenous glucocorticoid release, using the chicken as an experimental model. Endogenous glucocorticoid levels can be elevated by immunization with exogenous antigens or by injection of conditioned medium, e.g. supernatant of mitogen‐stimulated spleen cells. This effect is mediated by so‐called glucocorticoid‐increasing factors, and is considered to act as an immunoregulatory principle. Thymocyte DNA of so treated birds showed a typical “ladder” pattern after electrophoresis in a 1.8% agarose gel, and degradation could be prevented by RU 38486. This provides evidence that apoptosis can be induced by elevating endogenous corticosterone levels in vivo. By means of in situ nick translation (ISNT) and simultaneous immunofluorescence tests, it was possible to analyze various thymic subpopulations during apoptosis after treatment with exogenous glucocorticoids. Additionally, using confocal microscopical techniques, apoptosis of the same cells as analyzed by ISNT is shown. The possible role of elevated concentrations of endogenous glucocorticoids in regulating thymocyte cell death and autoimmune diseases is discussed.
T cell subset distribution and B cell hyperreactivity in mice expressing interleukin‐4 under the control of major histocompatibility complex class I regulatory sequences
Authors: Erb Klaus J. , Holtschke Thomas , Muth Kathrin , Horak Ivan , Schimpl Anneliese
Published: March 2007

Transgenic mice in which interleukin‐4 (IL‐4) is expressed under the control of the major histocompatibility complex (MHC) class I regulatory sequences show low level expression of IL‐4 in all organs investigated. Several weeks after birth the animals develop thymus hypoplasia with a loss of CD4+CD8+ double‐positive cells and a relative increase in the mature population, especially, and in contrast to previously published lines, the CD4+ single‐positive cells. In the periphery, T lymphocytes eventually decline, CD8+ cells being more strongly affected. Many of the residual T cells exhibit the CD44highMel‐14low phenotype of antigenically experienced T cells. B cells also show an activated phenotype with respect to size, MHC class II and CD23 expression, are more readily stimulated by anti‐μ F(ab′)2 antibodies than are B cells from control littermates, and show a higher spontaneous and antigen‐induced production of IgG1 and IgE.
Human anti‐IgE antibodies by repertoire cloning
Authors: Vogel Monique , Miescher Sylvia , Biaggi Christine , Stadler Beda M.
Published: March 2007

Anti‐IgE antibodies have been detected in sera of patients with an allergic disease where they might play a role in the regulation of (Ig)E‐mediated reactions. Using a recombinant phage surface display expression system a combinatorial library of antibody heavy and light chains was constructed from peripheral blood mononuclear cells from an atopic donor immunized with tetanus toxoid. Screening of the library allowed the identification of a large number of phages displaying human monovalent antigen‐binding fragments (Phab) against tetanus toxoid and IgE. Surprisingly, we found a high frequency of Phab against particular IgE myelomas that was comparable to the frequency found for Phab against tetanus toxoid. However, most of these Phab were directed to different idiotypic determinants, depending on the IgE myeloma used for the panning procedure. Nevertheless, two clones were found to have anti‐isotypic specificity and were shown to react specifically with the CH2 domain of the IgE heavy chain.
Differential contribution of thymic outputs and peripheral expansion in the development of peripheral T cell pools
Authors: Modigliani Yves , Coutinho Graça , Burlen‐Defranoux Odile , Coutinho Antonio , Bandeira Antonio
Published: March 2007

The number of peripheral T cells in mice increases up to 100‐fold in the first few weeks of life. We have followed the fate of Thy‐1 congenic T cells transferred into newborn recipients, to evaluate the relative contribution of thymic output versus peripheral expansion in the constitution of peripheral T cell pools during post‐natal development. The results show that in normal animals there is essentially no peripheral expansion of T cells, which show slow turnover rates (1 to 2 months) along that time period. The rates of cell accumulation in the periphery require, therefore, an average of 1 × 106−2 × 106 mature thymic emigrants/day for the first 3 weeks of life.
Effect of CTLA‐4 chimeric protein on rat autoimmune anti‐glomerular basement membrane glomerulonephritis
Authors: Nishikawa Kazuhiro , Linsley Peter S. , Collins A. Bernard , Stamenkovic Ivan , McCluskey Robert T. , Andres Giuseppe
Published: March 2007

The interaction of the T cell receptor with the antigen/major histocompatibility class II complex is insufficient to induce optimal T cell activation. Co‐stimulatory signals, including those provided by CD28/CTLA‐4 on T cells and B7 molecules (B7‐1, −2 and −3) on antigen‐presenting cells, are also required. CD28‐B7 interactions can be blocked by a soluble human CTLA‐4 chimeric protein (CTLA4Ig). We tested the effect of administration of CTLA4Ig on experimental anti‐glomerular basement membrane (GBM) autoimmune glomerulonephritis in Wistar‐Kyoto rats induced by immunization with bovine GBM. The disease is characterized by development of antibody to the α3 chain of type IV collagen (Goodpasture's antigen), deposition of rat IgG in GBM, infiltration of the kidney by T cells and macrophages, severe crescent formation and renal failure leading to death in 5–6 weeks. Animals injected with human CTLA4Ig from day 0 to day 14 or to day 35 had reduced disease severity. Beneficial effects were observed even when injections were begun after the onset of glomerulonephritis on day 14. However, the rats developed antibody to the human CTLA4Ig, associated with reduction in levels of circulating CTLA4Ig. The results provide evidence for CD28/CTLA‐4 signaling in rat autoimmune glomerulonephritis, and suggest that more effective inhibition of B7‐dependent T cell activation, such as might be achieved with homologous CTLA4Ig, could be useful in the treatment of autoimmune diseases.
Lipopolysaccharide‐induced cytokine production in human monocytes: Role of tyrosine phosphorylation in transmembrane signal transduction
Authors: Beaty Christopher D. , Franklin Terry L. , Uehara Yoshimasa , Wilson Christopher B.
Published: March 2007

The signal transduction events that follow the binding of lipopolysaccharide (LPS) to the macrophage cell surface are not well defined. In the current studies LPS was found to induce alterations in phosphorylation of monocyte proteins on tyrosine. Herbimycin A and genistein, inhibitors of tyrosine kinases, markedly attenuated LPS‐induced tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) protein and mRNA production. Reciprocally, the tyrosine phosphatase inhibitor sodium orthovanadate enhanced LPS‐induced production of TNF‐α. LPS induced a concentration‐dependent increase in tyrosine phosphorylation of several proteins, which paralleled and preceded the onset of LPS‐induced TNF‐α production. LPS stimulation had different but reproducible effects on three members of the src family of tyrosine kinases. Both Hck and Lyn kinase activity increased before the onset of TNF‐α production, consistent with their participation in the observed LPS‐induced tyrosine phosphoprotein accumulation. In contrast, Yes kinase activity was not affected. These observations were made at concentrations of LPS that required serum rich in LPS‐binding protein and the monocyte surface antigen CD14 for TNF‐α production. These data indicate that tyrosine kinases and phosphatases are involved in the signal transduction cascade by which LPS induces production of TNF‐α and IL‐6 by human monocytes, and suggest that Lyn and Hck are candidate participants in this process.
Cytokine synthesis and apoptosis by intestinal intraepithelial lymphocytes: Signaling of high density αβ T cell receptor+ and γδ T cell receptor+ T cells via T cell receptor‐CD3 complex results in interferon‐γ and interleukin‐5 production, while low density T cells undergo DNA fragmentation
Authors: Yamamoto Masafumi , Fujihashi Kohtaro , Amano Masahiko , McGhee Jerry R. , Beagley Kenneth W. , Kiyono Hiroshi
Published: March 2007

To study the biological consequences of cytokine production and apoptosis by intraepithelial lymphocytes (IEL), we have studied these characteristics in both the high and low density CD3+ IEL populations. Stimulation of low‐ or high‐density CD3+ IEL via the T cell receptor (TCR)‐CD3 complex using monoclonal anti‐CD3, anti‐αβ TCR or anti‐γδ TCR antibodies resulted in opposing effects. In one case, a significant number of the high‐density CD3+ T cells entered cell cycle from the resting stage (DNA replication was observed) and anti‐TCR‐CD3 treatment enhanced the numbers of interferon‐γ and interleukin‐5 spot‐forming cells in this cell fraction. In contrast, when the low‐density αβ TCR+ or γδ TCR+ T cells were activated via the TCR‐CD3 complex, DNA fragmentation was observed. These results demonstrated that the activation signals transduced via the TCR‐CD3 complex resulted in their entry into the cell cycle and subsequent interferon‐γ and interleukin‐5 production in the high‐density IEL T cell subset. However, identical signals induced apoptosis in the majority of the low‐density fraction of CD3+ IEL.
In vivo and in vitro IgE isotype switching in human B lymphocytes: Evidence for a predominantly direct IgM to IgE class switch program
Authors: Van Der Stoep Nienke , Korver Wouter , Logtenberg Ton
Published: March 2007

Molecular analysis of circular excision products and composite genomic switch regions has demonstrated that in mice, immunoglobulin (Ig) isotype switching from IgM to IgE often proceeds sequentially via IgG1. Based on analysis of Ig production in cell cultures, it has been suggested that human B cells may switch to IgE via IgG4, whereas limited molecular data from in vitro switched B cells suggest a direct IgM to IgE switch program. To obtain a quantitative assessment of direct versus sequential IgE switching in humans, we have analyzed the nucleotide sequences of 29 composite Sμ/Sϵ switch regions from freshly isolated human B lymphocytes from patients with atopic dermatitis and from B lymphocytes induced to switch to IgE synthesis in vitro. The data show that in these B cells IgE isotype switching progressed directly from IgM to IgE. We conclude that, in contrast to the murine IgM/IgE switch program, the IgM to IgE switch in B lymphocytes from patients with atopic dermatitis as well as in vitro stimulated B cells from healthy donors preferentially proceeds via direct Sμ to Sϵ switch recombination.
Pro‐T cells in fetal thymus express c‐kit and RAG‐2 but do not rearrange the gene encoding the T cell receptor β chain
Authors: Hozumi Katsuto , Kobori Akiko , Sato Takehito , Nozaki Hideki , Nishikawa Shin‐Ichi , Nishimura Takashi , Habu Sonoko
Published: March 2007

Ten percent of 15‐day fetal thymocytes of mice were Pgp‐1+Thy‐1lo cells. Half were strongly stained with monoclonal antibodies (mAb) recognizing the oncogene product, c‐kit, but were not stained with mAb against non‐T cell markers such as B220, Mac‐1 and Gr‐1. The isolated Pgp‐1+c‐kit+ thymocytes showed no rearranged bands for V‐DJ and D‐J of T cell receptor (TcR) β, but Pgp‐1−‐c‐kit− thymocytes showed D‐J rearranged bands. Both cells expressed the RAG‐2 gene which is required for the V(D)J recombination process. When Pgp‐1+c‐kit+ thymocytes were cultured in 2‐deoxyguanosine‐treated alymphocytic fetal thymus, they became TcR‐expressing mature type T cells, but this differentiation was reduced by the addition of anti c‐kit mAb. These data indicate that Pgp‐1+c‐kit+ thymocytes are pro‐T cells with the potential to differentiate mature T cells in the thymic environment. This study also indicates that c‐kit‐mediated signals promote the differentiation of thymocytes during their early stages.
Intracellular Ca2+ stores of T lymphocytes: Changes induced by in vitro and in vivo activation
Authors: Clementi Emilio , Martino Gianvito , Grimaldi Luigi M. E. , Brambilla Elena , Meldolesi Jacopo
Published: March 2007

Intracellular Ca2+ stores were investigated in resting and activated splenic T lymphocytes from Lewis rats. Activation was obtained either in vitro (spleen cells isolated from “naive” rats exposed to concanavalin A for 24 h) or in vivo (spleen cells from rats with fully developed symptoms of experimental allergic encephalomyelitis). In both experimental conditions several changes of Ca2+ homeostasis were observed with respect to resting lymphocytes: (1) a threefold increase of the total intracellular calcium (from 1.15 to 3.5 mmol/l); (2) a moderate increase of the pool sensitive to inositol 1,4,5‐trisphosphate (IP3), investigated both in intact T lymphocytes (fura‐2 and 45Ca2+‐release techniques in cells challenged with phytohemagglutinin) and in T lymphocytes permeabilized with β‐escin (45Ca2+ release induced by saturating concentrations of IP3); and (3) the appearance of a pool released by the endoplasmic reticulum (ER) Ca2+ ATPase inhibitor thapsigargin (Tg), but insensitive to IP3, which, therefore, appears to be localized in areas of the ER devoid of the cognate receptor. The latter two findings were paralleled in activated lymphocytes by an increase of expression of ER markers, involved (calreticulin; Ca2+ ATPase) or not (protein disulfide isomerase) in the regulation of Ca2+ homeostasis. In contrast, calnexin (another ER marker) and the receptor for IP3 were increased to only a moderate extent. Finally, an enlargement of non‐ER Ca2+ pools was observed in the cells pretreated with Tg in which 45Ca2+ release was induced by the Ca2+ ionophore ionomycin. Our results document structural and functional changes of intracellular Ca2+ stores which might play an important regulatory role in activated T lymphocytes.
Soluble transferrin mediates targeting of hepatitis B envelope antigen to transferrin receptor and its presentation by activated T cells
Authors: Gagliardi Maria‐Cristina , Nisini Roberto , Benvenuto Rosalba , De Petrillo Guido , Michel Marie‐Louise , Barnaba Vincenzo
Published: March 2007

In a previous study, we identified that transferrin receptor (TfR) is the receptor utilized by hepatitis B virus (HBV) to enter T cells. We demonstrated that hepatitis B envelope antigen (HBenv Ag) is taken up by activated T cells via TfR, processed in endosomal compartments, and presented on class II molecules to specific CD4+ T cell clones. Herein, we report that binding to soluble ferric Tf by HBenv Ag is needed in TfR‐mediated endocytosis. Accordingly, presentation of HBenvAg by activated T cells is not observed in serum‐free medium and is restored by addition of soluble Tf. Moreover, we provide evidence that pre‐S2 and S regions of HBenv Ag contain the critical residues for the interaction with soluble Tf. Our data not only explain HBV entry into a variety of host activated cells, but may also help in developing strategies to alter the course of chronic HBV infection.
Human CD45RA+ and CD45R0+ T cells exhibit similar CD3/T cell receptor‐mediated transmembrane signaling capacities but differ in response to co‐stimulatory signals
Authors: Schwinzer Reinhard , Siefken Renate , Franklin Richard A. , Saloga Joachim , Wonigeit Kurt , Gelfand Erwin W.
Published: March 2007

CD45RA+ cells have been described to be less responsive to CD3/T cell receptor (TcR)‐mediated activation than CD45R0+ T cells. To analyze the underlying mechanism of the differential responses we compared CD3/TcR‐triggered tyrosine phosphorylation in the two subsets and studied the role of co‐stimulatory signals provided either by accessory cells or pharmacologic activation of protein kinase C by phorbol ester. Stimulation of purified CD45RA+ and CD45R0+ T cells with CD3/TcR antibodies induced similar patterns and intensities of tyrosine phosphorylation in the two subsets, but no proliferation. If accessory cells were used as the source of co‐stimulatory signals, strong expression of the 55‐kDa chain of the interleukin‐2 (IL‐2) receptor (CD25), significant IL‐2 production and vigorous proliferation were observed in CD45R0+ cells, whereas CD45RA+ cells responded weakly. However, when CD3/TcR‐mediated triggering was combined with activation of protein kinase C by phorbol ester, CD45RA+ cells responded strongly. These data indicate that the transmembrane signaling capacity of the T cell receptor expressed by CD45RA+ and CD45R0+ cells is similar and, therefore, is presumably not responsible for the differential reactivities of the two subsets. It is more likely that co‐stimulatory signals determine whether CD3/TcR‐initiated activation results in strong or weak responses.
Interleukin‐13 alters the activation state of murine macrophages in vitro: Comparison with interleukin‐4 and interferon‐γ
Authors: Doyle Anthony G. , Herbein Georges , Montaner Luis J. , Minty Adrian J. , Caput Daniel , Ferrara Pascual , Gordon Siamon
Published: March 2007

Interleukin (IL)‐13 is a newly described cytokine expressed by activated lymphocytes. We examined the effects of the murine recombinant cytokine on the phenotype and activation status of elicited peritoneal macrophages (Mϕ), concentrating on activities which are known to be modulated by interferon‐γ and IL‐4. IL‐13 markedly suppressed nitric oxide release and to a lesser extent secretion of the pro‐inflammatory cytokine tumor necrosis factor‐α. However, antimicrobial capacity was not completely jeopardized as the respiratory burst was unaffected, and indeed the enhanced expression of Mϕ mannose receptor and major histocompatibility class II, and regulation of sialoadhesin, the Mϕ sialic acid‐specific receptor involved in hemopoietic and lymphoid interactions, suggest that these cells are not simply deactivated, but primed for an active role in immune and inflammatory responses. These activities closely mimic those of IL‐4, but mediation of the effects by IL‐4 was discounted by the use of a neutralizing monoclonal antibody. Thus, IL‐13, like IL‐4, is a cytokine which has complex effects on Mϕ behavior, inducing activities characteristic of both activation and deactivation.
The 5′ boundary of somatic hypermutation in a Vχ gene is in the leader intron
Authors: Rada Cristina , González‐Fernández Africa , Jarvis John M. , Milstein César
Published: March 2007

The maturation of the immune response involves the hypermutation of antibody genes and the selection of B cells expressing receptors with improved antigen binding properties. Somatic hypermutation of antibody genes is targeted to a small region ∼1 kb surrounding the rearranged V gene. The precise definition of the 5′ limit is not yet clear since the data base of somatic mutations upstream of the V region is very restricted. The available data suggest that it lies close to the promoter region and this has been used to implicate transcription in the mechanism leading to hypermutation. Here we present an extensive analysis of mutations in the 5′ region of a single χ light chain gene. A large data base from highly mutated sequences was obtained from anti‐oxazolone hybridomas expressing the VχOx1‐Jχ5 light chain and from polymerase chain reaction‐derived clones from splenic and Peyer's patches of transgenic mice expressing the same VχOx1‐Jχ5 gene combination. Although mutations were found in the 5′‐flanking segment, the rate of mutation in the V‐J segment was about 20‐fold higher. A sharp decline between those two mutation rates is evident but the boundary was found in the leader intron of the VχOx1 gene, about 150 bases downstream of the initiation of transcription site.
Induction of a cytotoxic T cell response by co‐injection of a T helper peptide and a cytotoxic T lymphocyte peptide in incomplete Freund's adjuvant (IFA): Further enhancement by pre‐injection of IFA alone
Authors: Valmori Danila , Romero Jackeline F. , Men Ying , Maryanski Janet L. , Romero Pedro , Corradin Giampietro
Published: March 2007

We have previously demonstrated that it is possible to induce a consistent and strong cytolytic T lymphocyte (CTL) response to synthetic peptides, corresponding to poorly immunogenic malaria CTL epitopes, by co‐injecting them with peptides representing defined T helper (Th) epitopes in incomplete Freund's adjuvant (IFA). In this study we have tested different immunization protocols to improve further the elicitation of the CTL response. We show that the CTL response to a mixture of Th + CTL peptides administered in IFA was further enhanced by a previous injection of the Th epitope peptide in IFA. Moreover, we found that the response could be significantly augmented by a pre‐injection of IFA alone. This enhancement was observed only if the Th epitope was also present in the second injection. The number of lymph node cells recovered was 2–3‐fold higher in mice pre‐injected with IFA, but the increase in specific CTL activity, expressed as lytic units per animal, by pre‐injection of IFA was at least 10–20‐fold. Thus, pre‐injection of IFA clearly increases the magnitude of a subsequent CTL response.
Epstein‐Barr virus (EBV) latent membrane protein 1 increases HLA class II expression in an EBV‐negative B cell line
Authors: Zhang Qin , Brooks Louise , Busson Pierre , Wang Fred , Charron Dominique , Kieff Eliott , Rickinson Alan B. , Tursz Thomas
Published: March 2007

Transformation of normal resting B cells by the Epstein‐Barr virus (EBV) leads to the establishment of permanent lymphoblastoid cell lines (LCL) which express high levels of HLA antigens and which are highly efficient in antigen presentation. Certain features of the LCL phenotype can be reproduced by transfecting EBV‐negative B lymphoma (BL) cell lines with individual EBV latent genes under heterologous promoters. In this work we have analyzed a series of subclones derived from the EBV‐negative cell line Louckes, stably transfected with constructs encoding EBV latent genes for their expression of HLA class II molecules. Louckes parental cells and control transfectants expressed detectable levels of HLA‐DR, DQ and DP antigens on the cellular surface by cytofluorometry, but these levels were significantly increased in transfectants expressing the virus‐coded latent membrane protein 1 (LMP‐1). Northern blotting for the individual α and β chain mRNA at each of the three HLA class II loci indicated correspondingly increased levels of HLA class II transcripts in the LMP‐1 transfectants. Transfectants expressing the virus‐coded nuclear antigens EBNA‐1, EBNA‐2 or EBNA‐LP showed no significant changes in these parameters. These observations indicate that up‐regulation of HLA‐class II molecules can be a part of the changes induced by LMP‐1 in B cells.
Induction of murine cytotoxic T lymphocytes against Plasmodium falciparum sporozoite surface protein 2
Authors: Wizel Benjamin , Rogers William O. , Houghten Richard A. , Lanar David E. , Tine John A. , Hoffman Stephen L.
Published: March 2007

Sporozoite surface protein 2 has been identified as a target of malaria vaccines designed to produce protective CD8+ cytotoxic T lymphocytes (CTL) because mice immunized with mastocytoma cells expressing a fragment of Plasmodium yoelii sporozoite surface protein 2 (PySSP2) are protected against malaria by an immune response that requires CD8+ CTL. To define CTL epitopes in the Plasmodium falciparum sporozoite surface protein 2 (PfSSP2), spleen cells (SC) from mice immunized with irradiated sporozoites (irr spz), were stimulated with synthetic peptides, and these effectors were tested for cytolytic activity against peptide‐pulsed, major histocompatibility complex (MHC)‐matched targets. Two peptides containing CTL epitopes, A6 (Pf SSP2 3D7 214–233) and BH1 (Pf SSP2 3D7 3–11) were identified in bulk cultures of SC from immune C57BL/6 mice, and by production of CTL lines. Immunization with recombinant vaccinia expressing the full length PfSSP2 induced antigen specific, MHC‐restricted, CD8+ T cell‐dependent cytolytic activity against these two peptides. Finally, CTL were induced by immunization with a bacteria‐derived recombinant fragment of PfSSP2 (rPfSSP2) mixed with a liposomal formulation containing a cationic lipid (Lipofectin® Reagent, LPF). Induced CTL lysed target cells pulsed with peptide A6 or with LPF/rPfSSP2, but not targets pulsed with only rPfSSP2. These studies demonstrate that CTL specific to PfSSP2 are present in C57BL/6 mice and that immunization with purified rPfSSP2 delivered with LPF induces a cytotoxic T cell response.
Inherited complement C3 deficiency: A defect in C3 secretion
Authors: Katz Yitzhak , Singer Lori , Wetsel Rick A. , Schlesinger Menachem , Fishelson Zvi
Published: March 2007

The molecular basis of inherited complement C3 deficiency in a 20‐year‐old newly diagnosed male patient was studied. Using an enzyme‐linked immunosorbent assay, the patient's C3 serum level was found to be approximately 7 μg/ml, which is less than 1 % of normal. In contrast, Northern analysis indicated that the patient's C3 mRNA was of normal size and quantity. Peripheral blood monocytes (PBM) and skin fibroblast cultures (F) from the patient and from healthy donors were labeled for 2 h with [35S] methionine. Analysis of cell lysates and supernatants by immunoprecipitation and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) demonstrated normal levels of C3 in lysates of patient's PBM and F. However, C3 secretion in the patient's cells was extremely reduced, with pulse‐chase experiments demonstrating a long delay in the disappearance of intracellular C3. Secretion of C1r and factor B by the patient's cells was normal. Lipopolysaccharide and interleukin‐1 increased C3 synthesis in the patient's PBM and F, but had no effect on the secretion. SDS‐PAGE analysis of trypsin‐cleaved intracellular C3 revealed an aberrant cleavage profile for the patient's C3. Collectively, these data indicate that C3 deficiency in this patient is due to a defect in the C3 secretion, probably as the result of abnormality in the proC3 structure.
Interleukin‐1‐induced activation of a protein kinase co‐precipitating with the type I interleukin‐1 receptor in T cells
Authors: Martin Michael , Böl Gaby Fleur , Eriksson Anders , Resch Klaus , Brigelius‐Flohé Regina
Published: March 2007

We have investigated the possibility of a protein kinase participating in the signal transduction mechanisms of the interleukin‐1 (IL‐1) type I receptor (IL‐1RI). Our data show that a protein kinase was co‐precipitated with the IL‐1 RI from the two murine T helper cell lines D10N and EL‐4. The kinase activity was detected in an in vitro kinase assay performed with the immuno beads in the presence of exogenous substrates. IL‐1 treatment of the cells resulted in a rapid activation of this protein kinase in a concentration‐dependent manner. Both forms of IL‐1, IL‐1α and IL‐1β, induced this kinase activity, whereas the IL‐1 receptor antagonist (IL‐1ra) was inactive. In excess IL‐1ra competitively antagonized IL‐1 stimulation. In the in vitro kinase assay the exogenous substrates myelin basic protein and histone H1 were phosphorylated, whereas casein or heat‐shock protein HSP27 were not accepted, reflecting a certain selectivity of this protein kinase. The IL‐1RI co‐precipitable protein kinase showed a serine/threonine specificity and was inhibited by staurosporine, but not by inhibitors specific for protein tyrosine kinases or protein kinase C. These results show that a serine/threonine protein kinase directly interacts with the IL‐1RI at the plasma membrane level of T helper cells forming a novel type of IL‐1 inducible signaling complex. This protein kinase may resemble the link coupling the plasma membrane IL‐1 receptor to cytosolic downstream elements in the IL‐1 signaling pathway.
Covalent binding of C3b to monoclonal antibodies selectively up‐regulates heavy chain epitope recognition by T cells
Authors: Santoro Lyse , Drouet Christian , Reboul Angeline , Mach Jean‐Pierre , Colomb Maurice G.
Published: March 2007

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b‐antigen complexes can bind to complement receptors on the antigenpresenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells.

In this study, IgG1,χ and IgG2a,χ murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and puritied in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared.

IgG or IgG‐C3b complexes presented by B cell lines were able to stimulate proliferation of χ light chain‐specific T cell clones at similar concentrations. In contrast, IgG‐C3b complex recognition by heavy chain‐specific T cell clones required 100‐fold less IgG‐C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigenpresenting cells.
Collagen‐specific cytotoxic T lymphocyte responses in patients with ankylosing spondylitis and reactive arthritis
Authors: Gao Xiao‐Ming , Wordsworth Paul , McMichael Andrew
Published: March 2007

Both ankylosing spondylitis (AS) and reactive arthritis (ReA) are strongly associated with HLA‐B27 although the mechanism for this association is still unknown. Here we examine the hypothesis that B27‐restricted, joint antigen‐specific cytotoxic T lymphocytes (CTL) may be the driving force of AS and ReA. Type II and type XI procollagens (CII and CXI, respectively), expressed almost exclusively in the articular cartilage of the joints, were chosen as the possible targets of autoimmune CTL. Type I procollagen (CI), expressed in many different tissues, was also included as control. Nineteen nonamer peptides bearing appropriate HLA‐B27 binding motifs from human CI, CII and CXI were identified and synthesized. When analyzed for binding affinity to HLA‐B27 in assembly assays, four (two from CII, two from CXI) were found capable of binding to HLA‐B27 with high affinity. These B27‐binding collagen peptides were then used to stimulate peripheral blood lymphocytes from eight B27‐positive AS and three ReA patients for identification of possible B27‐restricted autoimmune CTL. HLA‐B27‐restricted CTL specific for one of the CII peptides, P109 were found in one of the ReA patients, but in none of the others.
Structure of the gene coding for the human early lymphocyte activation antigen CD69: A C‐type lectin receptor evolutionarily related with the gene families of natural killer cell‐specific receptors
Authors: Santis Ana G. , López‐Cabrera Manuel , Hamann Jörg , Strauss Michael , Ánchez‐Madrid Francisco
Published: March 2007

CD69 is the earliest inducible cell surface glycoprotein acquired during lymphoid activation. CD69 functions as a signal transmitting receptor involved in cellular activation events including proliferation and the induction of specific genes. This molecule is a member of a supergene family of type‐II integral membrane proteins with C‐type lectin domains. We have herein studied the genomic structure of the human gene encoding CD69. The coding sequence is divided into five exons separated by four introns. The first two exons corresponded to separate functional domains of the protein (cytoplasmic tail and the transmembrane region), while the final three exons encoded the carbohydrate‐recognition domain (CRD). The conserved intron position between the exons encoding the CRD indicated that this protein is closely related to other type‐II receptor groups with the C‐type CRD, such as the asialoglycoprotein receptors, the low‐affinity IgE receptor (CD23), and natural killer cell‐specific receptors, NKR‐P1 and Ly49. In contrast to the broad NKR‐P1 and Ly‐49 gene families, CD69 is a single‐copy gene, as demonstrated by Southern blot analyses. The major transcription initiation site has been located, by amplification of cDNA 5′ ends, 30 nucleotides downstream of a consensus TATA box. Comparison of human CD69 and mouse NKR‐P1 gene structures indicates that the first intron maintains a conserved position, suggesting that CD69 and this gene family may diverge from a common ancestor gene. A possible evolutionary pathway of these genes is proposed.
Lymphadenopathy induced by the cooperation between lprcg and gld genes is of lpr but not of gld phenotype
Authors: Matsuzawa Akio , Katagiri Takuya , Ogata Yoshihisa , Kominami Ryo , Kimura Mikio
Published: March 2007

Mice homozygous for the lpr (lymphoproliferation), lprcg or gld (generalized lymphoproliferative disease) mutation develop strikingly similar lymphadenopathy with expansion of B220+ CD4− CD8− double‐negative (DN) T cells and autoimmunity. To elucidate the roles of bone marrow (BM) and lymph node (LN) in lymphoproliferation, BM and LN were transplanted simultaneously into normal or +/+ mice in various genotype combinations. In lpr/lpr or lprcg/lprcg BM recipients grafted lpr/lpr and lprcg/lprcg LN swelled but +/+ and gld/gld LN atrophied. In gld/gld BM recipients all of LN swelled regardless of genotype. Thus, lpr and lprcg are phenotypically different from gld in the interaction of BM‐derived DN T cells and +/+ LN. Compared with lpr the lprcg gene differs in its ability to complement with gld in induction of lymphadenopathy. To determine whether lymphoproliferation induced by the cooperation between lprcg and gld is of lpr or gld phenotype, LN of various genotypes were implanted into double heterozygous lprcg/+, gld/+ mice. Grafted lpr/lpr and lprcg/lprcg LN swelled but +/+ and gld/gld LN atrophied, indicating that it is of lpr phenotype. Moreover, grafted lprcg/+ LN swelled but lpr/+ LN atrophied, indicating that, in the heterozygous state, lprcg is phenotypically different from lpr as it allows for LN accumulation of DN T cells induced by lprcg‐gld cooperation.
A yeast artificial chromosome contig on mouse chromosome 11 encompassing the nu locus
Authors: Nehls Michael , Lüno Karsten , Schorpp Michael , Krause Sabine , Matysiak‐Scholze Uta , Prokop Christa‐Maria , Hedrich Hans J. , Boehm Thomas
Published: March 2007

Mutations at the nude locus disrupt the homing process of T cell progenitor cells to the thymic rudiment, a key aspect of T cell differentiation. Here, we map the nude locus to a set of overlapping yeast artificial chromosomes (YAC) clones covering a genetic interval of about 0.5 centi Morgan on mouse chromosome 11. These results provide a suitable starting point to molecularly clone the nude gene.
Interleukins (IL)‐1 and IL‐2 control IL‐2 receptor α and β expression in immature thymocytes
Authors: Wilsonu Anne , Corthésyu Patricia , Reichenbachu Patrick , MacDonaldu H. Robson , Nabholz Markus
Published: March 2007

Functional high‐affinity interleukin‐2 receptors (IL‐2R) contain three transmembrane proteins, IL‐2Rα, β and γ. We have investigated the expression of IL‐2Rα and β genes in immature mouse thymocytes. Previous work has shown that during differentiation these cells transiently express IL‐2Rα on their surface. Stimulation of IL‐2Rα+ and IL‐2Rα− immature thymocytes with phorbol 12‐myristate 13‐acetate and calcium ionophore induces synthesis of IL‐2Rα and IL‐2Rβ mRNA. Most of this response depends on autocrine stimulation by IL‐2. IL‐1 synergizes with IL‐2 to induce a 120‐fold increase in IL‐2Rα mRNA and a 14‐fold increase in IL‐2Rβ mRNA levels. A large proportion of the stimulated cells contains both transcripts. These interleukins do not induce any differentiation to more mature phenotypes. Collectively, these results show that IL‐2 plays a major role in the regulation of IL‐2R expression in normal immature thymocytes. We suggest that this response to interleukins may be part of a homeostatic mechanism to increase the production of immature thymocytes during stress.
New infectious mammary tumor virus superantigen with Vβ‐specificity identical to staphylococcal enterotoxin B (SEB)
Authors: Lutheru Sanjiv , Shakhov Alexander N. , Xenariosu Ioannis , Hagau Satomi , Imaiu Shunsuke , Acha‐Orbea Hans
Published: March 2007

Only few infectious mouse mammary tumor viruses (MMTV) have been characterized which induce a potent superantigen response in vivo. Here we describe the characterization of an MMTV which was isolated from milk of the highly mammary tumor‐prone SHN mouse strain. Exposure of newborn mice to milk‐borne MMTV (SHN) results in a very slow deletion of Vβ7, 8.1, 8.2 and 8.3 expressing peripheral T cells. Subcutaneous injection of adult mice with this virus induces a rapid and strong stimulation of all four affected Vβ‐subsets in vivo. Besides the strong T cell effect we observed an early proliferation and activation of the local B cell pool leading to the initial secretion of IgM followed by preferential secretion of IgG2a, by day 6. Sequence comparison of the polymorphic C terminus with known open reading frames revealed high homology to the endogenous provirus Mtv‐RCS. This is the first report of a virus having a complete overlap in Vβ‐specificity with a bacterial superantigen stimulating as many as 35 % of the whole CD4+ T cell repertoire including Vβ8.2.
Local and systemic consequences of acute, low‐dose ultraviolet B radiation are mediated by different immune regulatory mechanisms
Authors: Shimizuu Tadamichi , Streilein J. Wayne
Published: March 2007

Acute, low‐dose ultraviolet B (UVB) radiation alters the local skin site such that epicutaneous application of hapten fails to induce contact hypersensitivity (CH), but induces tolerance in UVB‐susceptible mice. Although the inability of irradiated skin to support CH induction may be a strictly local effect, there may also be systemic immune consequences of UVB radiation delivered in this manner. To examine this matter, abdominal skin of C57BL/6 mice was exposed to acute, low‐dose UVB radiation. Dinitrofluorobenzene was immediately painted directly on the irradiated site, or at a distant (unirradiated) site. In separate experiments, epicutaneous application of the hapten on a distant site was delayed for 1–3 days. The mice were tested for acquisition of CH, and for tolerance, i.e. the capacity to become sensitized when exposed subsequently to hapten via normal body wall skin. It was found that, immediately after completion of the UVB regimen, CH was inducible via unirradiated, but not via irradiated, skin. At 3 days post‐UVB exposure, CH was no longer inducible even through unirradiated skin. Mice that first encountered hapten via UVB‐exposed skin developed tolerance, as did mice that first encountered hapten via unirradiated skin of UVB‐treated mice. Neutralizing anti‐tumor necrosis factor‐α TNF‐α antibodies failed (a) to restore the ability of unirradiated skin to support induction of CH, and (b) to interfere with tolerance induction, whether hapten was first painted on irradiated or unirradiates skin. The data indicate that the acute, low‐dose regimen of UVB radiation produces effects on the immune system that are manifest locally as well as systemically. By demonstrating that the disruption of CH induction following UVB radiation is TNF‐α dependent, whereas locally and systemically induced tolerance is not, our findings encourage further search for other UVB‐related modulators of systemic immunity and tolerance.
Variable region gene usage of human monoclonal rheumatoid factors derived from healthy donors following immunization
Authors: Thompson Keith M. , Randen Ingrid , Børretzen Marie , Førre Øystein , Natvig Jacob B.
Published: March 2007

A study was performed to compare the use of immunoglobulin V gene segments by rheumatoid factors (RF) produced in physiological responses following a defined antigenic stimulus, with RF produced in rheumatoid arthritis (RA) and RF produced as monoclonal (M)‐components in certain lympho‐proliferative diseases. A panel of 46 monoclonal RF was produced, using hybridoma techniques, from healthy individuals following immunization with foreign antigens (mis‐matched red blood cells). A panel of previously characterized monoclonal RF from RA synovial tissues was extended to a total of 24 and included in the study. The variable heavy (VH) and variable light (VL) chain gene families used by these RF were determined using idiotypic markers and polymerase chain reaction amplification with VH‐specific primers. The frequencies of expression of the various gene families was compared between the two groups, and compared with the published expression frequencies seen amongst M‐component RF. The majority (87 %) of RF from healthy donors were found with light chains using V gene segments of the Vχ3 family, in conjunction with VH gene segments belonging to the VH1, VH3 and VH4 families. The over‐expression of Vχ3, together with the distribution of VH families, demonstrates close similarities with RF found as M‐components in lympho‐proliferative diseases. In contrast, RF from RA patients showed a predominant use of VH3 gene segments (82 %) and an unbiased expression of Vχ3 segments (29 % of the light chains). These data suggest that RF found as M‐components are representative of RF used in normal physiological responses, but have undergone neoplastic or other transformation. RF found in the synovial tissue of RA patients appear to be driven by different mechanisms than RF seen in physiological responses in healthy individuals.