bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of T cell receptor γ chains from adult CD4‐CD8‐ thymocytes
Authors: Moore Mark W. , Crispe I. Nicholas , Bevan Michael J.
Published: March 2007

The role of T cell receptor (TcR) γ genes in T cell development has not been determined. To extend our understanding of the repertoire of TcR γ expression, we prepared a cDNA library from CD4‐CD8‐ adult BALB/c thymocytes and cloned and sequenced 15 TcR γ genes from this cDNA library. We found that two clones were transcripts of the unrearranged Cγ2 gene and that three clones terminated in the Jγ2 region. Nine of the remaining clones were Vγ1.2Jγ2Cγ2 genes and five of these were in frame. Only one clone corresponded to Cγ1 and resulted from an in‐frame Vγ2Jγ1Cγ1 join. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the γ chain proteins from the surface of both BALB/c and C57BL/6 adult CD4‐CD8‐ thymocytes did not detect the 32‐kDa Vγ1.2Cγ2 protein, but did detect the 35‐kDa VγCγ1 protein. These results suggest that despite the abundance of full‐length functional Vγ1.2Cγ2 transcripts in the thymocyte subset, the protein product is not expressed on the cell surface as the predicted 32‐kDa γ protein. Finally, our analysis of the V‐J joining of the γ genes reveals both flexibility at the V‐J junction and extensive N‐region nucleotide addition that lead to diversity of the predicted protein sequence.
Species specificity of interleukin 2 binding to individual receptor components
Authors: Collins Mary K. L.
Published: March 2007

The affinity of recombinant human and murine interleukin 2 (IL 2), which differ significantly in structure in the amino terminal α helix, for the human or murine interleukin 2 receptors was compared. Murine IL 2 showed a 10‐fold higher affinity than human IL 2 for the murine high‐affinity receptor expressed on T lymphocytes, whereas human IL 2 bound 100‐fold more strongly than murine IL 2 to the human high‐affinity receptor. As the high‐affinity IL 2 receptor on T lymphocytes has been demonstrated to be composed of two IL 2 binding components, p55 and p75, the role of the individual chains in the determination of species specificity was then investigated. Recombinant retroviral vectors were used to generate populations of 3T3 fibroblasts expressing either human or murine p55 in isolation, and their binding characteristics were determined. While the murine p55 showed an identical species preference to the murine p55/p75 high‐affinity complex, the human p55 had an equal affinity for human and murine IL 2. This suggested that the human p75 chain was responsible for the preference for human IL 2 shown by the human high‐affinity receptor, which was confirmed by performing a binding experiment using gibbon MLA 144 cells, expressing only p75. These data suggest that the amino terminal region of the IL 2 molecule interacts with the p75 chain of the IL 2 receptor.
Tolerance induction in the organ‐cultured thymus lobes upon intimate contact with allogeneic thymus lobes
Authors: Suzuki Gen , Kawase Yoshiko , Hirokawa Katsuiku
Published: March 2007

In an organ‐cultured murine fetus thymus, precursor cytotoxic T lymphocytes (pCTL) specific for alloantigens developed successfully but those reactive with self antigens were eliminated. In attempting to dissect the mechanism of self tolerance, intrathymic chimera was made by culturing two genetically disparate thymuses in close contact with each other (parabiosis of thymuses). This maneuver resulted in the induction of specific and mutual CTL tolerance. It seems that CTL tolerance was induced by clonal deletion but not by active suppression. Since 2'‐deoxyguanosine treatment abolished the tolerogenic capability of the thymus, hemopoietic cells capable of migrating to and fro in the parabiotic thymuses are thought to be responsible for tolerance induction. Induction of CTL tolerance was dependent on the maturation stages of T cells in the thymus: T cells in 5‐day‐, but not in 7‐day‐cultured thymuses were susceptible to tolerance induction, indicating that T cells expressing T cell receptors at low density are susceptible to tolerance induction.
Non‐random expression of T cell receptor γ and δ variable gene segments in functional T lymphocyte clones from human peripheral blood
Authors: Borst Jannie , Wicherink Annemieke , Dongen Jacques J. M. Van , Vries Evert De , Comans‐Bitter W. Marieke , Wassenaar Fred , Elsen Peter Van Den
Published: March 2007

Human T cell receptor (TcR) γδ displays a variety of protein forms. Disulfide‐linked (type 1) or non disulfide‐linked (type 2) receptors occur, with γ chains encoded by the Cγ1 or the Cγ2 gene segment, respectively. Exon 2 of Cγ2 may either be duplicated or triplicated (type 2a or 2b receptors). TcR γ chains differ in molecular mass and charge between type 1 and type 2 receptors. The δ chains as well as the γ chains have different structural properties between receptor types. This cannot be due to the use of different Cδ gene segments, since the genome encodes only one. To understand the genetic basis of this dichotomy in γ/δ combinations, rearrangement and expression of Vγ, Jγ, Cγ and Vδ gene segments were determined in TcR γ/δ+ clones derived randomly from peripheral blood of normal donors. Most clones used Cγ1, a minority Cγ2. The different protein properties of receptor types could be explained by the nonrandom expression of Vγ(Jγ) and Vδ gene segments. Type 1 receptors preferentially used γ chains encoded by the Vγ9 and Jγ1.2 gene segments together with δ chains encoded by Vδ2. In type 2a receptors, Vγ9 was not predominant; often other Vγ gene segments were employed, but then in high frequency in coordination with Vδ1. Reactivity of the clones with monoclonal antibodies anti‐TiγA, BB3 and δ‐TCS‐1 correlated with the expression of the Vγ9, Vδ2 and Vδ1 gene segments, respectively. Therefore, Vγ and Vδ use in TcR γ/δ+ cells from peripheral blood of eight healthy individuals, including the two donors of the clones, could be determined tentatively by double immunofluorescence. Indeed, the Vγ9‐Vδ2 combination was predominant, while the Vγ9‐Vδ1 and particularly the Vγ9‐“Vδother” combination was rare. These data indicate that the TcR γδ repertoire in peripheral blood of normal individuals is largely dependent on junctional diversity and suggest that selection of receptors occurs.
Expression and function of HLA‐A2.1 in transgenic mice
Authors: Epstein Helen , Hardy Robert , May Janet S. , Johnson Martin H. , Holmes Nicholas
Published: March 2007

We have derived a number of transgenic mouse lines which express the human major histocompatibility complex class I gene HLA‐A2.1. Two lines carry the complete human HLA‐A2.1, the others bear a recombinant gene in which the HLA‐A2.1 coding regions are fused to the H‐2Kb promoter. Analysis of transgenic spleen cells by immunofluorescence demonstrates that these mouse cells express HLA‐A2.1 on their surface in association with mouse β2‐microglobulin (β2m), confirming that HLA‐A2 does not require human β2m to be expressed at the cell surface. The cells contain more HLA mRNA than endogenous H‐2 class I mRNA. There is also a large pool of non‐β2m‐associated HLA heavy chain inside the cell. In contrast the amount of HLA: β2m complex is low. Thus, in transgenic mice HLA‐A2 seems to compete poorly with H‐2 heavy chains for mouse β2m.

The HLA‐A2.1 transgenic mice do not produce influenza‐virus‐specific cytotoxic T cells (CTL) restricted to the HLA transgene, at least in sufficient numbers to be measured in a direct bulk CTL assay. The dominance of H‐2‐restricted clones may be the result of quantitative rather than qualitative factors. However, HLA‐A2.1 transgenic spleen cells are effective in stimulating an allogeneic CTL response in normal mice. This response is not H‐2 restricted. Cold target inhibition studies show that there are at least two populations of CTL, one of which is specific for HLA‐A2.1 on mouse cells. This result suggests that at least some allo‐CTL are directed against major histocompatibility complex plus “self‐peptide”.
The effects of bacterial lipopolysaccharide, anti‐receptor antibodies and recombinant interferon on mouse B cell cycle progression using 5‐bromo‐2'‐deoxyuridine/hoechst 33258 dye flow cytometry
Authors: Seyschab Helga , Friedl Richard , Schindler Detlev , Hoehn Holger , Rabinovitch Peter S. , Chen Una
Published: March 2007

Polyclonal stimulation of resting B cells with anti‐antigen receptor antibodies [anti‐IgM μ chain antibody (anti‐μ)] or with bacterial lipopolysaccharide (LPS) stimulates a proportion of B cells to proliferate. Exposure of resting B cells to both LPS and anti‐μ activates a larger population of resting B cells than either alone, suggesting a synergistic effect of these two stimuli. Although recombinant interferon (rIFN) either alone or in combination with anti‐μ has no apparent proliferative activity (as measured by [3H]thymidine incorporation), application of 5‐bromo‐2'‐deoxyuridine/Hoechst 33258 dye flow cytometry reveals a distinct effect of rIFN on B cell growth. In the presence of anti‐μ plus LPS, rIFN causes the majority of B cells to enter the cell cycle (CC), but a subset of B cells remains in the resting stage. Another subset of B cells has extremely rapid CC transit times, with a CC duration of less than 10 h. These studies show that both anti‐μ and LPS are competence factors (which move cells from the G0 phase to the G1 phase). LPS acts also as a CC progression factor, while rIFN is a CC potentiating factor.
The molecular basis of the low hemolytic activity of C4 molecules from low‐C4 mice with IgM‐coated erythrocytes
Authors: Dieli Francesco , Sireci Guido , Lio Domenico , Salerno Alfredo
Published: March 2007

This study investigated the origin of the different hemolytic activity of two allotypes of murine C4, C4H (C4‐high) and C4L (C4‐low) in the presence of IgM‐coated erythrocytes. C4H displayed a threefold higher hemolytic titer (expressed in hemolytic units/μg protein) than C4L. No difference was found between C4H and C4L either in stability at 37 °C at different pH values and in the rate of C4H and C4L hydrolysis by activated C1.

The major functional difference was found in the covalent binding capacity to IgM‐coated erythrocytes, with the amount of C4H bound being about threefold higher than that of C4L. A marked difference in the reactivity of the C4b fragment of C4H and C4L toward amino and hydroxyl groups was detected. Using glycine and glucose to competitively inhibit the binding of C4H and C4L, it was observed that C4L selects preferentially for the amino groups of glycine, whereas C4H reacted preferentially with the hydroxyl groups of glucose. Furthermore, the chemical modification of the red cells amino groups by ethylacetimidate, had no appreciable effect on the binding of C4H, but reduced that of C4L. Southern blot hybridization experiments using Hind III‐digested liver DNA and a cDNA probe derived from the 5' end of the mRNA (clone pAT‐A) indicated that the C4H allele was associated with a 5‐kb Hind III fragment, whereas the C4L allele was associated with a 15‐kb Hind III fragment. The possibility that a structural difference between C4H and C4L is responsible for their different reactivities is discussed.
Visualizing interleukin 2 gene expression at the single cell level
Authors: Emilie Dominique , Peuchmaur Michel , Barad Marc , Jouin HéLèNe , Maillot Marie‐Christine , Couez Dominique , Nicolas Jean‐FrançCois , Malissen Bernard
Published: March 2007

To analyze the expression of the interleukin (IL)2 gene at the single cell level, we have constructed a chimeric gene in which the regulatory sequences from the mouse IL 2 gene were fused 5' proximal to the coding region of the Escherichia coli β‐galactosidase (lacZ) gene. Once stably introduced into a T cell hybridoma, the IL 2‐lacZ reporter gene was shown to display a pattern of induction similar to the one observed for the resident IL 2 gene. Two points emerged from monitoring IL 2 expression at the single cell level. First, upon activation the expression of the IL 2‐lacZ gene appears asynchronous. Second, at the peak of induction the percentage of β‐galactosidase+ cells never reached 100% and the level of β‐galactosidase reached among positive cells was highly heterogeneous within a cloned population of T cells. In combination with the possibility to sort viable lymphocytes according to lacZ expression, the IL 2‐lacZ reporter gene described herein should provide a way to isolate somatic cell mutants deficient in signal transduction by the T cell antigen receptor complex.
Allosuppression of B cells in vitro by graft‐vs.‐host reaction‐derived T cells is caused by cytotoxic T lymphocytes
Authors: Rozendaal Lawrence , Pals Steven T. , Schilham Marco , Melief Cornelis J. M. , Gleichmann Ernst
Published: March 2007

An acute graft‐vs.‐host reaction (GVHR) was induced by i.v. injection of 108 lymphoid cells from C57BL/10 (B10) donors (H‐2b/b) into adult non‐irradiated (B10 x DBA/2)F1 mice (H‐2b/d). Previous experiments have established that spleen cells obtained from such GVHF1 mice suppress the primary antibody response of normal F1 spleen cells to sheep red blood cells. This type of suppression was termed “allosuppression”, and it was shown to be mediated by Ly‐2+ T cells of donor origin that react against H‐2 antigens of the host. It was unclear, however, whether the actual mechanism of allosuppression was due to suppressive factors generated by donor T cells or whether the latter killed the F1 B cells. Here, we show that for their suppressive effect GVHF1 spleen cells need direct cell contact with F1 spleen cells; no suppressive effect was measured in a double‐chamber culture system in which the GVHF1 spleen cells were separated from the responding normal F1 spleen cells by a cell‐tight membrane filter. The suppressive effect only affected cells expressing the appropriate H‐2 class I alloantigen; in mixed cultures of irradiated F1 spleen cells and GVHF1 spleen cells with third‐party B cells the antibody response of the third‐party B cells was not suppressed. GVHF1 spleen cells showed cytotoxic T lymphocyte (CTL) activity specific for the allogeneic F1 H‐2 antigen. The suppressive effect of the GVHF1 spleen cells did not differ from that exerted by cloned CTL specific for MHC class I alloantigens; cloned CTL suppressed the primary antibody response of murine spleen cells without affecting the response of third‐party B cells added to the cultures. The combined findings show that “allosuppression” in vitro is not due to suppression of F1 B cells, but to a direct killing of these cells by alloreactive CTL.
Immunoglobulin‐specific T‐B cell interaction
Authors: Rudensky Alexander Y. , Yurin Vitalij L.
Published: March 2007

Immunoglobulin (Ig)‐specific T‐B cell interactions have been studied in the model of T cell recognition of the χ chain Igχ‐1b allotype in Igχ‐1‐congeneic rat strains. An efficient presentation of endogenous Ig allotypic determinants by irradiated spleen cells from (WAG.1b x August)F1 (RT‐1u/c; Igχ‐1b/1a) rats to Igχ;‐1b‐specific lymph node T cells from Igχ;‐1‐congeneic (WAG x August)F1 (RT‐1u/c; Igχ‐1a) rats was demonstrated. This presentation was found to be sensitive to high irradiation doses (WAG x 1000 rad). By fractionation of Igχ‐1b+ F1 spleen cells on Percoll density gradient we have shown that a radioresistant, low‐density fraction, consisting mainly of macrophages (MΦ) and dendritic cells, triggers only weak Igχ;‐1b‐specific T cell response. The high level of response was observed against radiosensitive spleen cell fractions of intermediate and high density, suggesting that B cells were the main antigen‐presenting cells (APC) of Igχ;‐1b determinants of endogenous Ig. This conclusion was confirmed in the experiments using purified B cells from Igχ;‐1b‐bearing rats. Earlier we have shown that the responsiveness of August (RT‐1c; Igχ;‐1a) and WAG (RT‐1u; Igχ;‐1a) rats to Igχ;‐1b in vivo is controlled by the dominant allele of an RT‐1‐linked Ir gene. August and (August x WAG)F1 rats were found to be responders to Igχ;‐1b while WAG rats were nonresponders. The same pattern of Ir gene‐controlled reactivity was demonstrated using an Igχ;‐1b‐specific T cell proliferation assay. Igχ;‐1b‐specific F1 T cell response was only observed when Igχ;‐1b+ B cells or IgG (Igχ;‐1b)‐pulsed Mϕ‐bearing responder major histocompatibility complex (MHC) haplotype were used as the APC. Anti‐RT‐1 monoclonal antibody inhibition studies suggested that the RT‐1Bc molecule is the main restricting element of T cell recognition of Igχ;‐1b+ B cell as well as exogenous IgG (Igχ;‐1b). We have demonstrated allelic exclusion of Igχ;‐1b presenting function by negatively and positively selecting for Igχ;‐1b+ and Igχ;‐1a+ B cells from heterozygous F1(Igχ;‐1b/la) rats. This clearly indicate that the B cells presented exclusively Igx‐1b allotypic determinants of their own Ig. Furthermore, it was found that Igχ;‐1b‐specific T cell activation was not caused by the in vitro transfer of Ig(Igχ;‐1b) molecules from one B cell to another, or to some residual APC of another type. Thus, B cells presented with high efficiency Igχ;‐1b determinants of self‐synthesized Ig in association with MHC class II molecule to Igχ;‐1b‐specific T cells.
The heterogeneity of the reticulum of rat peripheral lymphoid organs identified by monoclonal antibodies
Authors: Berg Timo K. Van Den , Döpp Ed A. , Brevé John J. P. , Kraal Georg , Dijkstra Christine D.
Published: March 2007

We have developed a panel of six monoclonal antibodies, ED10–ED15, directed against reticular cells in peripheral lymphoid organs. Immunohistochemistry revealed prominent differences between these antibodies with regard to their tissue distribution in lymphoid and non‐lymphoid organs. Furthermore, the determinants recognized by ED10–ED13 were found to be differentially expressed by reticular cells occupying the various specialized compartments present in peripheral lymphoid organs. The reactivity patterns of these antibodies observed during the ontogenetic development of the spleen suggest that they recognize differentiation antigens expressed by reticular cells. In contrast, ED14 and ED15 were found to have a relatively ubiquitous tissue distribution recognizing reticular cells in each compartment, with a constitutive reactivity during splenic ontogeny. The present results indicate that reticular cells form a heterogeneous population within the lymphoid organs.
B cell repertoire in adult antigen‐free and conventional neonatal balb/c mice
Authors: Bos Nicolaas A. , Meeuwsen Cornelia G.
Published: March 2007

Early in ontogeny B cells preferentially use VH gene families which are most adjacent to the genes coding for the constant part of the immunoglobulin molecule. In conventional adult mice, however, a random usage of VH gene families has been found. We investigated the role of exogenous antigenic stimulation on this normalization of VH gene usage by B cells. Therefore, we made use of adult germ‐free BALB/c mice fed a chemically defined ultrafiltered antigen‐free diet (GF‐CD) and neonatal conventional BALB/c mice. Both the adult GF‐CD and the newborn conventional mice represent situations with minimal exogenous antigenic stimulation. The results obtained with RNA dot blot hybridization with probes specific for the different VH gene families showed in hybridomas from adult GF‐CD BALB/c mice a preferential usage of the CH‐proximal VH gene family PC7183. In hybridomas from 5‐day‐old conventional BALB/c mice a less frequent usage of the J558 VH gene family was found and an increased usage of the PC7183 VH gene family than what would be expected from random usage. Evidence is presented that the RNA giving a positive signal with the PC7183 probe represents functional messages for IgM production.
The restrictive role of sialic acid in antigen presentation to a subset of human peripheral cd4+ t lymphocytes that requires antigen‐presenting dendritic cells
Authors: Kapsenberg Martien L. , Stiekema Frank E. M. , Kallan Aram , Bos Jan D. , Roozemond Robert C.
Published: March 2007

Dendritic cells from human epidermis, i.e., Langerhans cells (LC), are more potent antigen‐presenting cells (APC) than APC from peripheral blood in proliferative in vitro responses of helper T lymphocytes to various soluble antigens. Analysis of antigen recognition by CD4+ T lymphocyte clones indicated that this increased potency of LC as APC results from a preferential requirement for LC of part of the T cell population. These T cells show a long‐lasting restoration of antigen responsiveness to peripheral blood APC after antigen‐specific restimulation in vitro, indicating that restrictive antigen recognition concerns T cells that are not fully differentiated. A similar restrictive antigen recognition was observed by treatment of the T cells or the APC with neuraminidase. This restoration of responsiveness is associated with the occurrence of nonspecific cell clustering between T cells and APC. These results suggest that the selective requirement for APC is regulated by the function of adhesion molecules that are functionally blocked by sialic acid groups on immature peripheral T cells but that are readily available on peripheral T cells at a later stage of differentiation.
Extracellular but not intracellular calcium mobilization is required for epstein‐barr virus‐containing supernatant‐induced b cell activation
Authors: Dugas Bernard , Mencia‐Huerta Jean‐Michel , Braquet Pierre , Galanaud Pierre , Delfraissy Jean‐FrançOis
Published: March 2007

Changes in intracytosolic free calcium concentration ([Ca2+]i) are thought to be an important trigger for the initiation of the cellular events culminating in B cell activation. After exposure of human B lymphocytes loaded with the fluorescent indicator quin‐2 to the Epstein‐Barr virus (EBV)‐containing supernatant of B95‐8 cell line, a rise in [Ca2+]i is observed. To determine the respective contribution of the intra‐ and extracellular Ca2+ pools in EBV‐induced B cell activation, the pharmacologic modulation of these processes was investigated using an intracellular calcium chelator, 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic (BAPTA) or the calcium channel blocking drugs. When the extracellular Ca2+ contribution was minimized in the presence of calcium channel‐blocking drugs or incubation of the cells in Ca2+‐free medium, the EBV‐induced human B cell activation was fully inhibited. When the calcium channel‐blocking drugs, either verapamil or diltiazem, were withdrawn or when exogenous Ca2+ was added to the Ca2+‐free medium, EBV‐induced B cell activation was noted, demonstrating the reversibility of the inhibition. On the contrary, when the intracellular Ca2+ contribution was reduced after loading the cell with BAPTA, no alteration of the EBV‐induced B cell activation was observed. Thus, the EBV‐induced rise of [Ca2+]i required in the activation of human B cells appears to be essentially related to the entry of extracellular Ca2+ and not to the release of Ca2+ from internal stores.
Isolation and characterization of ige receptors from rat intestinal mucosal mast cells
Authors: Swieter Mark , Chan Bosco M. C. , Rimmer Carol , Mcneill Karol , Froese Arnold , Befus Dean
Published: March 2007

High‐(FcϵRI) and low‐(FcϵRII) affinity IgE receptors were isolated from surface radioiodinated, Nonidet‐P40‐solubilized rat intestinal mucosal mast cells (IMMC) and compared with those on rat peritoneal mast cells (PMC) and rat basophilic leukemia (RBL) cells. FcϵRII were isolated by affinity chromatography using IgE‐Sepharose or by anti‐FcϵRII antisera and protein A‐Sepharose. The surface‐exposed, IgE‐binding α subunits of FcϵRI [FcϵRIα] were isolated by affinity chromatography using IgE and anti‐IgE‐Sepharose. FcϵRIα on IMMC had an apparent molecular mass of 59 kDa, somewhat larger than that of PMC (51 kDa), RBL‐2H3 cells (51 kDa) or RBL‐CA10.7 cells (46 kDa). Brief (45 s) incubation of IMMC or PMC in glycine‐HCl, pH 3, prior to iodination removed much of the surface‐bound IgE. This permitted more thorough labeling of the receptors, but had no affect on the estimate of receptor size. Surprisingly and in contrast to acid‐treated PMC, upon anti‐IgE‐Sepharose isolation acid‐treated IMMC yielded an intensely radioactive FcϵRIα band in the absence of added IgE. Such a finding suggests that IMMC, more so than PMC, may have an intracellular store of IgE, as has been suggested by many others. IMMC also differed from PMC in the number of forms of FcϵRII isolated; 50‐kDa and 58‐kDa forms of FcϵRII were obtained from IMMC, whereas PMC yielded most often a single 56‐kDa FcϵRII band. These results were mimicked by the two RBL cell sublines: RBL‐2H3 cells yielded two FcϵRII (46 kDa and 55 kDa), but only one form of FcϵRII (54‐kDa) was obtained from RBL‐CA10.7 cells. Thus, the two subtypes of rat mast cells, which have previously been shown to differ in mediator profile and responsiveness to secretagogues and antiallergic drugs, are also distinguished by differences in IgER profile.
The proliferation and life‐span of rat large granular lymphocytes: effects of cytokines
Authors: Vaage John Torgils , Reynolds Craig W. , Reynolds Della , Fossum Sigbjørn , Rolstad Bent
Published: March 2007

Pulse labeling of large granular lymphocytes (LGL) with [3H]thymidine for 1 h in vitro showed that 1%–7% of LGL were in S phase in the blood, spleen and liver of unstimulated euthymic and athymic rats as scored with autoradiography. Repetitive injections of [3H]thymidine over 2–7 days revealed that about half of the blood and spleen LGL had formed by division of precursors during this week. Stimulation of rats with the interferon inducer poly(I) · poly(C) increased the proportion of S phase LGL rapidly and simultaneously in the blood, spleen and liver, so that by 20 h after stimulation ca 30% of LGL were in the S phase in these organs. This LGL proliferation was accompanied by an increased number of LGL in all three compartments 48–96 h after poly(I) · poly(C) injection. Blood LGL cultured in cell impermeable diffusion chambers in the peritoneal cavity of poly(I) · poly(C)‐stimulated rats exhibited enhanced natural killer (NK) activity but no proliferative response, indicating that mature LGL were not induced to undergo blastogenesis by poly(I) · poly(C) in vivo. Pretreatment of NK cells with rat interferon (IFN‐α/β) and with human recombinant interleukin 2 (rIL2) in vitro, showed that these two cytokines, when combined, had opposing effects on NK activity and proliferation: whereas rIL2 inhibited the IFN‐induced augmentation in NK activity, IFN inhibited the rIL2‐induced LGL proliferation.
Dna fragmentation and cell death mediated by t cell antigen receptor/cd3 complex on a leukemia t cell line*
Authors: Takahashi Shuji , Maecker Holden T. , Levy Ronald
Published: March 2007

An anti‐T cell receptor (TcR) monoclonal antibody (mAb), LC4, directed against a human leukemic T cell line, SUP‐T13, caused DNA fragmentation (“apoptosis”) and cell death upon binding to this cell line. Cross‐linking of receptor molecules was necessary for this effect since F(ab')2, but not Fab', fragments of LC4 could induce cell death. Five anti‐CD3 mAb tested also caused apoptosis, but only when they were presented on a solid phase. Interestingly, soluble anti‐CD3 mAb induced calcium flux and had an additive effect on the calcium flux and interleukin 2 receptor expression induced by LC4, but these anti‐CD3 mAb reversed the growth inhibition and apoptosis caused by LC4. The calcium ionophore A23187, but not the protein kinase C activator phorbol 12‐myristate 13‐acetate (PMA), also induced apoptosis, suggesting that protein kinase C activation alone does not cause apoptosis, although PMA is growth inhibitory. These results suggest that two distinct biological phenomena can accompany stimulation of the TcR/CD3 complex. In both cases, calcium flux and interleukin 2 receptor expression is induced, but only in one case is apoptosis and cell death seen. The signal initiating apoptosis can be selectively prevented by binding CD3 portion of the receptor in this cell line. This difference in signals mediated by the TcR/CD3 complex may be important in explaining the process of thymic selection, as well as in choosing anti‐TcR mAb for therapeutic use.
Different large deletions of t cell receptor vβ genes in natural populations of mice*
Authors: Jouvin‐Marche Evelyne , Trede Nikolaus S. , Bandeira António , Tomas Alfredo , Loh Dennis Y. , Cazenave Pierre‐André
Published: March 2007

A panel of geographically separate Mus m. domesticus and Swiss mice from several sources was screened for deletions in the T cell receptor variable (V) β locus. Four out of forty‐three strains tested show a deletion identical to or larger than the deletion previously described in SJL mice. To our knowledge, this is the first time that such important Vβ deletions are described in inbred or partially inbred wild‐derived strains of mice. On the other hand there seems to be very little polymorphism between the remaining Vβ genes. Expression of Vβ genes in peripheral and intra‐thymic T cells was tested using antibodies specific for different Vβ polypeptide chains. Flow cytometry analysis revealed a high expression of Vβ6 and Vβ17 genes in the Copacabana Swiss‐derived strain and an absence of Vβ17 expression in the WLA wild‐derived strain.

The three Mus m. domesticus strains (WLA, DDO and WBG) having deleted two to three additional Vβ subfamilies compared to SJL present no apparent immune deficiencies or autoimmune disorders. We conclude that relatively few Vβ genes may suffice for unimpaired survival of wild mice and that there is a selective pressure for the structural conservation of the remaining Vβ genes.
T cell receptor expression on immature thymocytes with in vivo and in vitro precursor potential
Authors: Nikolic‐Zugic Janko , Moore Mark W.
Published: March 2007

Immature CD8‐CD4‐ double‐negative (DN) thymocytes differentiate intrathymically into CD8+CD4+ and CD8‐CD4+ thymocytes and migrate to the periphery. This differentiation proceeds through several intermediate phenotypic changes in the expression of CD8 and CD4. We have recently established the existence of a CD8loCD4lo cell population in murine thymus that can repopulate the irradiated thymus in vivo and differentiate rapidly in vitro to CD8+CD4+ double‐positive (DP) cells. The CD8loCD4lo cells score as DN upon direct cytofluorometric analysis, yet are distinct from true DN cells by various criteria. Experimental evidence strongly suggests that they are descendants of true DN in the maturation pathway. In the experiments presented here, we further characterize this CD8loCD4lo thymocyte population. Northern blot and RNA protection analysis reveal that these cells transcribe full length mRNA for the T cell receptor (TcR)α chain, unlike the less mature interleukin 2 receptor‐positive DN thymocytes. Surface expression of the TcR‐associated CD3 molecule occurs on ∼ 15% of these cells at low levels characteristic of immature cells. In the course of in vitro differentiation a vast majority (∼ 80%) of these cells convert to CD8+CD4+ and significant numbers of the brightly staining DP convertants (11%–34% on day 1 and 48%–68% on day 2) express immature levels of CD3. Our results indicate that CD8lo, CD4lo cells might be the first thymic subset to rearrange TcR α chain genes and express TcR α/β heterodimer on the surface at levels characteristic of immature cells. Furthermore, the surface expression of TcR persists on the in vitro progeny of these thymocytes.
T cell activation: independent induction of killing activity and interleukin 2 secretion in cytolytic hybridomas*
Authors: Ozery Tali , Berke Gideon , Moscovich Miriam , Ozato Keiko , Kaufmann Yael
Published: March 2007

Memory‐like cytotoxic T lymphocytes (CTL) hybridomas exhibiting inducible killing activity and IL2 production were used to analyze the anamnestic response of CTL. Four activating agents were examined; anti‐Thy‐1 monoclonal antibody G7, staphylococcal enterotoxin B, interferon (IFN)‐α/β and IFN‐γ. These agents seemed to affect CTL activities in three distinct ways. Anti‐Thy‐1 monoclonal antibody, like specific antigen, was found to be a potent inducer of specific killing and IL2 production, whereas staphylococcal enterotoxin B induced IL2 production, but not cytolytic activity. On the other hand, IFN‐α/β and IFN‐γ effectively stimulated cytotoxicity without inducing IL2 production. The independent triggering of specific killing and IL2 secretion in the monoclonal cytolytic hybridomas suggests that in CTL distinct signals stimulate killing activity and IL2 production. The results also suggest that IFN‐α/β and IFN‐γ trigger the cytolytic program through an alternative activation pathway which does not involve the T cell receptor.
The lymphocyte‐specific protein tyrosine kinase p56lck is hyperphosphorylated on serine and tyrosine residues within minutes after activation via T cell receptor or CD2
Authors: Danielian Silvia , Fagard Remi , Alcover AndrÉS , Acuto Oreste , Fischer Siegmund
Published: March 2007

Human T cells can be activated and induced to proliferate through either the antigen‐specific receptor complex (TcR‐CD3) or the CD2 surface molecule. Following stimulation, both serine and tyrosine phosphorylation of cellular protein have been demonstrated to occur. p56lck, a protein tyrosine kinase associated to the inner face of the plasma membrane, is almost exclusively expressed in lymphoid cells, especially T cells. Within minutes after activation of a human T cell‐derived line (Jurkat) via stimulation of either the TcR‐CD3 complex or the CD2 glycoprotein, we observed a hyperphorphosylation of p56lck. A concomitant shift to a higher molecular weight in sodium dodecyl sulfatepolyacrylamide gel was also observed. Similar changes were obtained with phorbol 12‐myristate 13‐acetate. Tryptic phosphopeptide analysis of the hyperphosphorylated form of p56lck yielded new phosphorylated sites in serine residues and an increased tyrosine phosphorylation. These results suggest that p56lck may be intimately connected to the signaling pathway in T cell activation.
In vivo administration of interleukin 1 elicits increased Ia antigen expression on B cells through the induction of interleukin 4
Authors: Killar Loran M. , Hatfield Cheryl A. , Carding Simon R. , Pan Mary , Winterrowd Gregory E. , Bottomly Kim
Published: March 2007

The effects of the in vivo administration of interleukin 1 (IL 1) on lymphocytes from lymph node and spleen were analyzed. Mice received five daily subcutaneous (s.c.) injections of various doses of human recombinant IL 1β. Either 1 or 7 days after IL 1 treatment, spleens, popliteal and inguinal lymph nodes were collected. Lymphadenosis and splenomegaly were observed in the IL 1‐treated animals. Lymph nodes from IL 1‐treated mice contained a higher percentage of B cells than controls, and B cells from IL 1‐treated mice expressed dramatically increased levels of Ia antigen. Lymphadenosis and splenomegaly, as well as the changes in subset distributions and Ia expression were transient. Concomitant treatment of mice with IL 1 and anti‐IL 4 monoclonal antibody suppressed IL 1 effects on B cell Ia expression, but not on the B/T cell ratio. In situ hybridization analyses revealed that IL 1 treatment induced the expression of mRNA for IL 4, interferon‐γ, and IL 2 in lymph node and spleen cells. The distribution of cells expressing the various cytokine mRNA was markedly different between the spleens and lymph nodes.
Expression and function of CD2 during murine thymocyt
Authors: Yagita Hideo , Asakawa Jun‐Ichi , Tansyo Shigeru , Nakamura Tetsuya , Habu Sonoko , Okumura Ko
Published: March 2007

CD2, originally recognized as the sheep erythrocyte receptor of human T cells, has been implicated in early T cell development in the thymus. In this report, we examined the expression and functional role of CD2 during murine thymocyte ontogeny by using monoclonal antibodies to murine CD2. Surface expression of CD2 was first detected in Thy−1+ fetal thymocytes at day 14 of gestation and it progressively increased during CD4−CD8− phenotype. Surface IL 2 receptor (CD25) expression was readily detected in surface CD2− cells at day 13 of gestation and the majority of CD2+ cells appeared to be generated from CD25+ cells thereafter. In adult CD4−CD8− thymocytes, the expression of CD2 and CD25 was mutually exclusive. These results indicate that surface CD2 expression is not a prerequisite for CD25 induction during murine thymocyte ontogeny. This was further confirmed by fetal thymus organ culture in which anti‐murine CD2 mAb was included. The antibody treatment led to a suppressed CD2 expression on thymocytes; however, there was no effect on the appearance of CD25. Moreover, no influence on the development of mature CD3+ thymocytes was observed after fetal thymus organ culture in the presence of anti‐murine CD2 mAb, and a substantial number of CD3+CD2− cells was demonstrated in fetal and adult CD4−CD8− thymocytes. These findings argue against the functional relevance of CD2 expression during early T cell development as proposed in humans.
Immunological alterations inducible by mercury compounds. III. H‐2A acts as an immune response and H‐2E as an immune “suppression” locus for HgCl2‐induced antinucleolar autoantibodies
Authors: Mirtcheva Jasmina , Pfeiffer Christiane , Bruijn Jan Anthonie De , Jacquesmart FrÉDÉRic , Gleichmann Ernst
Published: March 2007

In responder mouse strains repeated injections of subtoxic doses of HgCl2 induce formation of antinuclear autoantibodies (ANA) and antinucleolar autoantibodies (ANolA). Others have shown that responsiveness to HgCl2‐induced formation of ANA and ANolA is linked to H‐2. Here, we extend these studies to a variety of mouse strains not tested previously. After confirming that strain B10.S (H‐2s) is a high responder we have shown that strains B10.D2 (H‐2d) and B10.BR (H‐2k) are nonresponders. By comparing a panel of strains carrying appropriate intra‐H‐2 recombinant haplotypes derived from d, k and s, we were able to map responsiveness to As. Interestingly, among four strains all of which were As, and thus responsive, only the two H‐2E‐ ones, B10.S and B10.RSD2, were high responders whereas the two H‐2E+ ones, B10.HTT and B10.S(9R), were significantly less responsive. Thus, the genetics of HgCl2‐induced autoantibody formation follow the rules established for immune responses to a variety of different antigens in that expression of H‐2E “suppressed” the response.
Recombinant interleukin (IL) 2‐induced human B cell differentiation is mediated by autocrine IL6
Authors: Xia Xing , Lee Hern‐Ku , Clark Steven C. , Choi Yong Sung
Published: March 2007

The molecular mechanism of the interleukin (IL) 2‐induced differentiation of human B cells has been investigated. The experimental results show that Staphylococcus aureus Cowan strain I (SAC) activation alone induces IL 6 secretion from B cells. When B cells were activated by SAC, there was an increased transcription of the IL 6 mRNA. It reached the peak level by 6 h and rapidly decreased to an undetectable level within 24 h. The IL 6 concentration in the culture supernatants reached the peak at 24–48 h and decreased slightly in the following culture periods. Since IL 2 alone could induce IgG secretion, whether exogenous IL 6 was added or not, and IL 2 did not increase autocrine IL 6 synthesis, it appears that IL 2 induces the IL 6 responsiveness of SAC‐activated B cells to differentiate in the later stage of the culture. The addition of polyclonal anti‐IL 6 antibody inhibited IgG secretion. The antibody still efficiently blocked IgG secretion up to day 5, indicating an important role of autocrine IL 6 in the IL 2‐driven B cell differentiation. However, the saturation dose of anti‐IL 6 antibody inhibited 50%–70% of IgG secretion, suggesting that IL 2‐induced B cell differentiation appears to be mediated by other factors besides IL 6.
Serum immunoglobulin levels and naturally occurring antibodies against carbohydrate antigens in germ‐free BALB/c mice fed chemically defined ultrafiltered diet
Authors: Bos Nicolaas A. , Kimura Hirohisa , Meeuwsen Cornelia G. , Visser Hendrik De , Hazenberg Maarten P. , Wostmann Bernard S. , Pleasants Julian R. , Benner Robbert , Marcus Donald M.
Published: March 2007

This study investigates the influence of exogenous antigenic stimulation on the serum immunoglobulin levels and the levels of circulating natural antibodies against carbohydrate antigens. Thus, BALB/c mice, raised in a germ‐free environment and fed a chemically defined, ultrafiltered diet (GF‐CD), were employed. These mice had normal serum IgM levels, but IgG and IgA levels were approximately 5% of conventionally reared littermates. The concentrations of all four IgG isotypes were equally low. The variable part of the heavy chains of naturally occurring BALB/c antibodies against a number of carbohydrate antigens, including 3‐fucosyllactosamine (3‐FL), levan and dextran, are encoded by VH441, and these antibodies express cross‐reactive idiotopes recognized by the monoclonal antibodies 6C4 and 6B1. Antibodies against levan and dextran were lower in GF‐CD than in conventional mice, but levels of anti‐3FL antibodies, and 6C4 and 6B1 idiotopes, were comparable to those in conventional animals. Peptidoglycan polysaccharide complexes (PPC) are carbohydrate antigens of bacterial origin, like levan and galactan. Naturally occurring antibodies against PPC were found in the serum of conventional mice, but were severely reduced in GF‐CD mice. The results indicate that most naturally occurring antibodies against carbohydrate antigens of bacterial origin found in conventional mice are caused by exogenous stimulation.
Identification by sequence analysis of chemotactic factors for monocytes produced by normal and transformed cells stimulated with virus, double‐stranded RNA or cytokine
Authors: Damme Jo Van , Decock Benny , Lenaerts Jean‐Pierre , Conings René , Bertini Riccardo , Mantovani Alberto , Billiau Alfons
Published: March 2007

A monocyte chemotactic activity was found to be released by various types of cultured human cells after appropriate stimulation: normal diploid fibroblasts, peripheral blood mononuclear cells or monocytes isolated therefrom, and a number of tumor cell lines, including osteosarcoma (MG‐63) and hepatoma (Malavu) but not melanoma (Bowes) cells. Cultures of diploid human fibroblasts and these tumor cells stimulated with interleukin (IL) 1 or double‐stranded RNA [poly(rI)·poly(rC)], or infected with viruses (measles or rubella viruses) were found to produce chemotactic activity for both monocytes and granulocytes. Media collected from fibroblasts treated with E. coli or IL 6 did not contain such activity.

Granulocyte and monocyte chemotactic activities were serologically distinct, and could be separated by successive chromatographical procedures. While the granulocyte chemotactic activity of both fibroblasts and MG‐63 cells had previously been identified as granulocyte chemotactic protein/IL 8, the monocyte chemotactic activity from MG‐63 cells was identified by amino acid sequence analysis as a different protein recently described to be released by human glioma and myelomonocytic cell lines. In view of the similarity in their chromatographical behavior, monocyte chemotactic activities from fibroblasts, MG‐63 cells and fresh monocytes can probably be assigned to identical molecules. Cultures of unfractionated peripheral blood cells, however, were found to release an additional monocyte chemotactic protein, identifiable by amino acid sequence analysis as platelet factor 4.
Human interleukin 2 (IL 2) receptor β chain allows transduction of IL 2‐induced proliferation signal(s) in a murine cell line
Authors: Doi Takeshi , Hatakeyama Masanori , Minamoto Seijiro , Kono Takeshi , Mori Hisashi , Taniguchi Tadatsugu
Published: March 2007

Interleukin 2 (IL 2) delivers cell growth signal by virtue of its interaction with the high‐affinity receptor complex, which consists of two distinct IL 2‐binding molecules: the IL 2 receptor α (IL 2R α) and β (IL 2R β) chains. Unlike many known growth factor receptors, neither of the IL 2R chains seems to contain a tyrosine kinase domain. In this report, we have shown that the human IL 2R β chain expressed in a murine IL 3‐dependent, non‐lymphoid cell line can transduce IL 2‐induced cell proliferation signal(s) in combination with the autologous mouse IL 2R α chain. This observation should provide a tool to dissect IL 2‐induced signal transduction pathway in lymphoid and non‐lymphoid cells.
Localization of interleukin 6 mRNA in human tonsils by in situ hybridization
Authors: Bosseloir Alain , Hooghe‐Peters Elisabeth L. , Heinen Ernst , Cormann Nadine , Kinet‐Denoël CÉCile , Vanhaelst Luc , Simar LÉOn
Published: March 2007

We have investigated which areas produce interleukin 6 (IL 6) in human tonsils. This growth factor is required for the terminal differentiation of B lymphocytes into plasmocytes. Using 35S‐labeled IL 6 cDNA we demonstrated IL 6 gene expression over various areas of the tonsils, with consistent exception of the follicles, by in situ hybridization. It is, therefore, proposed that B cells are stimulated during their migration out of the follicles.
Accessory factors involved in murine T cell activation. Distinct roles of interleukin 6, interleukin 1 and tumor necrosis factor
Authors: Vink Anne , Uyttenhove Catherine , Wauters Pierre , van Snick Jacques
Published: March 2007

Interleukin (IL) 6 was compared to other macrophage‐derived products for its capacity to support the proliferation of accessory cell‐depleted T cells. Monoclonal anti‐IL 6 antibodies were found to inhibit completely the “accessory activity” of macrophage supernatants, thus demonstrating the central role played by IL 6 in T cell activation. IL 6 was apparently more critical for initiating than in maintaining T cell proliferation because anti‐IL 6 antibodies lost all inhibitory activity when added late to the culture. Moreover, IL 6 was not the only accessory factor required for optimal T cell proliferation. Using low‐density cultures to minimize the number of contaminating accessory cells, we found that significant proliferation of CD4 cells was obtained only in the presence of both IL 6 and IL 1. In contrast, with CD8 cells substantial proliferation was obtained with IL 6 alone. This response could, however, be enhanced by IL 1. Tumor necrosis factor (TNF) and granulocyte/macrophage colony‐stimulating factor showed no activity in these assays. The concentrations of IL 1 and of IL 6 required to support optimal proliferation were 10 pg/ml and 1 ng/ml, respectively.

Analysis of the mechanisms underlying T cell activation by IL 1 and IL 6 indicated that both cytokines were required for optimal production of IL 2 but that IL 6 alone was sufficient to confer IL 2 responsiveness. For CD8 cells, this effect was observed with doses of IL 6 about 100 times lower than those required for the induction of IL 2 secretion (0.001 vs. 0.1 ng/ml). TNF, which was not capable of inducing IL 2 secretion, was also found to induce IL2 responsiveness but only at a concentration ≈ 1000 times higher than that of IL 6. In accordance with these observations, IL 6 and to a lesser extent TNF were found to enhance IL 2R expression by CD8 cells. Interestingly, this enhancing effect was totally dependent on the presence of IL 2.