bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of murine leukemia viruses on the function of dendritic cells
Authors: Gabrilovich Dmitry I. , Roberts Mary S. , Harvey Jennifer J. , Botcherby Marc , Bedford Penelope A. , Knight Stella C.
Published: March 2007

In asymptomatic human immunodeficiency virus‐1 infection T cells respond normally to allogeneic dendritic cells (DC), but DC show reduced stimulatory capacity. By contrast in HTLV‐1 infection no significant changes in allogeneic stimulation were seen but DC‐stimulated activity of autologous T cells. In seeking animal models relevant to these diseases the effects of two murine leukemia retroviruses, Rauscher leukemia virus (RLV) and Moloney leukemia virus (MLV) on the function of dendritic cells and T cells in a primary mixed leucocyte reaction have been tested. Treatment by RLV in vitro suppressed the ability of DC to stimulate allogeneic T cells from healthy animals. MLV at the same concentration did not significantly affect the ability of DC to stimulate allogeneic T cells, but provoked considerable enhancement of the low level stimulation by DC in the syngeneic system. Similar results were obtained following in vivo exposure to viruses. Two pieces of evidence suggested that these effects were due to impairment of DC function and were not operating through infection of T cells. Firstly, exposure of T cells directly to virus in vitro and in vivo before stimulation with untreated allogeneic DC caused no significant alteration in T cell activity. Secondly, the impact of murine leukemia virus on DC function was not abrogated when infected DC were added to normal T cells and cultured in the presence of zidovudine. Treatment of DC by RLV caused a decrease of cluster formation with allogeneic T cells. No statistically significant influence of MLV was observed on cluster formation after 3‐h of incubation in the allogeneic system. However, after 18‐h incubation MLV‐treated DC formed fewer clusters with T cells than untreated DC. At the same time a stimulatory effect of MLV on DC cluster formation with syngeneic T cells was found. Considerable decrease was found in major histocompatibility complex class II antigen and LFA‐1 receptor expression on the DC surface in mice infected by RLV MLV induced no significant changes. These mouse retroviruses can therefore cause changes in DC function similar to those already reported using human retroviruses and may provide models for studying their effects.
Cleavage and inactivation of human C1 inhibitor by the human leukocyte proteinase, proteinase 3
Authors: Leid R. Wes , Ballieux Bart E. P. B. , van der Heijden Ineke , Kleyburg‐van der Keur Carin , Hagen E. Christiaan , van Es Leendert A. , van der Woude Fokko J. , Daha Mohamed R.
Published: March 2007

Incubation of highly purified human C1 inhibitor with equally pure human leukocyte proteinase 3, resulted in a dose‐ and time‐dependent inactivation of C1 inhibitor hemolytic activity. Furthermore, this inactivation was accompanied by proteinase 3‐dependent cleavage of the C1 inhibitor into an 83 000 molecular weight fragment. The formation of the 83 000 molecular weight fragment followed a time course which was similar to that observed for the inactivation of hemolytic activity. Within 120 minutes more than 90% of the hemolytic activity was lost. This inactivation of C1 inhibitor appeared to be selective as purified human Clq was not degraded in a similar time period. Moreover, when 100 μg IgG, isolated from each of 21 Wegener's granulomatosis patients with cytoplasmic anti‐nuclear antibodies immunofluoresent titers to proteinase 3 greater then 1:64, was incubated with 3 milliunits of proteinase 3, little to no cleavage of C1 inhibitor was observed. In contrast, 100 μg of IgG isolated from 14 normal donors was ineffective in affording protection to C1 inhibitor upon incubation with proteinase 3. Our results suggest that neutrophil infiltration and activation could lead to local complement consumption at the tissue sites.
Selection of the expressed B cell repertoire by infusion of normal immunoglobulin G in a patient with autoimmune thyroiditis
Authors: Dietrich Gilles , Varela Francisco J. , Hurez Vincent , Bouanani Majida , Kazatchkine Michel D.
Published: March 2007

In the present study we have analyzed the changes in the expressed antibody repertoire and in temporal fluctuations of antibody levels in serum that followed infusion of normal IgG (IVIg) in a patient with autoimmune thyroiditis. Administration of IVIg resulted in the stimulation of IgM production, in alterations of expressed antibody activity in serum that could not merely be accounted for by the passive transfer of antibody specificities contained in IVIg, in transient down‐regulation of B cells clones expressing a specific disease‐related idiotype and in the increase in serum in recipient's autoantibodies specifically reactive with F(ab′)2 fragments of IVIg. In addition, infusion of IVIg shifted the pattern of spontaneous fluctuations of autoantibody activities in the patient's serum from a pattern indicative of disconnected events in the immune network to a pattern similar to that which is consistently observed in healthy controls. These results suggest that normal IgG may modulate autoreactivity by selecting expressed antibody repertoire through V region‐dependent interactions with antibodies.
Interleukin‐1 dysregulation is an intrinsic defect in macrophages from MRL autoimmune‐prone mice
Authors: Levine Jerrold S. , Pugh Barbara J. , Hartwell Daqing , Fitzpatrick John M. , Marshak‐Rothstein Ann , Beller David I.
Published: March 2007

Macrophages (Mϕ) from pre‐diseased autoimmune‐prone MRL mice (both MRL/+ and MRL/1pr) dramatically underproduce the cytokine interleukin‐1 (IL‐1) in comparison to Mϕ from a number of normal strains. In this study we show that IL‐1 dysregulation by MRL Mϕ is fully expressed at birth, and that this defect does not change with time or the development of disease. We also constructed adult irradiation chimeras (consisting of A/J → MRL and MRL → A/J mice), and show that Mϕ isolated from these chimeras display a pattern of IL‐1 production indistinguishable from that of the donor strain controls. Moreover, when we constructed a mixed chimera (A/J + MRL → A/J), the A/J and MRL Mϕ coexisting within the same animal retained their individual patterns of IL‐1 production when isolated by negative selection. Taken together, these results provide the first substantive evidence for an intrinsic defect (IL‐1 dysregulation) in Mϕ from MRL autoimmune‐prone mice.
Autoimmunity, hyporeactivity to T cell mitogens and lymphoproliferative disorders following neonatal induction of transplantation tolerance in mice
Authors: Jánossy Tamás , Baranyi Lajos , Knulst André C. , Vizier Csaba , Benner Robbert , Kelényi Gábor , Végh Pál
Published: March 2007

We have reported that, in A/J (A) (H‐2a) mice, a partial tolerance to C57BL/10ScSn (B10) (H‐2b) skin allografts and a high incidence of lethal lymphoproliferative disorders (LPD) can be induced by the neonatal i.v. injection of 2 × 107 semiallogeneic (B10 × A)F1 spleen cells (SC) (Végh, P., Baranyi, L. and Jánossy, T., Cell. Immunol. 1990. 129: 56). In this study, we show that the incidence and mortality of LPD were continuously growing from 1 month of age until the end of the experiment at 1 year (64% and 36%, respectively). Based on histology, 27% of the diseased mice suffered from lymphoid malignancies. In the remaining cases (73%), reactive histopathological changes were seen in the spleen, lymph nodes (LN), liver and kidneys. The proportion of CD4+Tcells in the spleen and LN as well as that of splenic B cells decreased, while the percentages of mature and immature myeloid cells doubled. The total cell number of each (sub)population, however, was elevated in both lymphoid organs. The cells taking part in the lymphoproliferation were of host (A) and not of donor (F1) origin. Preceding the development of apparent LPD, the SC, LN cell and thymus cell suspensions of 1‐month‐old tolerized mice showed reduced in vitro proliferative responses to cell and T cell‐dependent B cell mitogens (Con A or PWM), while their reactivity to aT cell‐independent B cell mitogen (lipopolysaccharide) was essentially unimpaired. This hyporeactivity seems to be functional, because neither histology nor immunophenotyping by flow cytometry revealed significant alterations in the spleen and thymus of such animals, apart from a slight reduction in the ratio of CD4+/CD8+ T cell subpopulations in the spleen. The in vivo T cell‐mediated immune response of the tolerized mice was practically normal to third party CBA/Ca (H‐2k) allografts. Antithymocyte autoantibodies (ATA) were detected in the sera of 76% of the tolerized mice at 1 month of age (i.e., even before the mass appearence of LPD). ATA as well as antinuclear Ab were present in 65% of the adult tolerized mice, independently of the presence of LPD. Taken together, in A mice neonatally injected with (B10 × A)F1SC, a partial, specific allograft tolerance and a chronic host‐vs.‐graft disease‐like syndrome developed. The latter is manifested in hyporeactivity to T cell mitogens, development of autoantibodies and, subsequently, in progressive LPD and lymphoid malignancies.
Antigens pre‐bound to the primate erythrocyte complement receptor via cross‐linked bispecific monoclonal antibody heteropolymers are rapidly cleared from the circulation
Authors: Reist Craig J. , Combs Matthew J. , Croft Barbara Y. , Taylor Ronald P.
Published: March 2007

We have used cross‐linked, bispecific monoclonal antibody heteropolymers, specific for primate erythrocyte (E) complement receptor (CR1) and target antigen (Ag), to attach 125I‐labeled Ag to 51Cr‐labeled monkey erythrocytes (E) in vitro. Injection of these sensitized E into monkeys leads to rapid clearance from the circulation of several different 125I‐labeled Ag with little, if any sequestration, lysis, or clearance of the E. It should now be possible, in principle, to use the heteropolymer‐E system to facilitate the clearance of blood‐borne pathogens from the circulation.
Peptide variants reveal how antibodies recognize major histocompatibility complex class I
Authors: Hogquist K. A. , Grandea A. G. , Bevan M. J.
Published: March 2007

The T cell receptor (TcR) on CD8+ T lymphocytes recognizes a complex which consists of a major histocompatibility complex (MHC) heavy chain, β2‐microglobulin (β2M), and peptide on the surface of antigen‐presenting cells. Mutational analyses have suggested that the TcR recognizes both the αl and α2 domains of the heavy chain as well as the peptide. In light of this, it is of interest to know to what extent the heavy chain domains take on distinct conformations when bound to individual peptides.

It has recently been shown that antibodies which recognize the Kb MHC complex are sensitive to which peptides are bound in the groove. We have extended this analysis to include eight Kb‐specific antibodies, seven of which are peptide sensitive. These antibodies, all of which are allo‐antibodies, recognize Kb‐bearing cells which, it is now appreciated, have a highly heterogeneous mix of self peptides presented in their grooves. We show that these self peptides also can affect antibody binding.

It has been suggested that peptides alter the conformation of the αl and α2 domains of the heavy chain and that this in turn affects the recognition of Kb by antibody. An alternative hypothesis is that solvent‐exposed peptide side chains may prevent the antibody from binding the complex. Using a panel of 128 single‐amino acid variants of a Kb‐binding antigenic peptide from ovalbumin we show that for most Kb‐specific antibodies, the second idea is more likely. Those variants which prevent antibody binding are at solvent exposed positions, and in general, the bulkier the side chain, the greater the inhibition of antibody binding. However, in the case of two antibodies, 100.30 and 34.4.20, the peptide residues which affect antibody recognition are buried, suggesting that these antibodies see an alternate conformation of the peptide/MHC complex.
No independent association between a tumor necrosis factor‐α promotor region polymorphism and insulin‐dependent diabetes mellitus
Authors: Pociot Flemming , Wilson Anthony G. , Nerup Jørn , Duff Gordon W.
Published: March 2007

Several studies have implicated tumor necrosis factor (TNF)‐α in the pathogenesis of insulin‐dependent diabetes mellitus (IDDM). In the present study we analyzed the first reported TNF‐α gene polymorphism in relation to IDDM. We have made frequence analysis and tested in vitro lipopolysaccharide (LPS)‐induced TNF‐α secretion. A significant difference in allele frequency was observed between patients and controls (p = 0.03). However, a very strong association of the uncommonTNF2 allele was observed with the HLA‐B8, –DR3 alleles. The relative risk (RR) of TNF2 was 2.2 compared to a RRof 3.1 for DR3. One reason for this difference was the identification of the TNF1 allele on the otherwise strongly IDDM‐associated HLA‐DR3 haplotype: DQB1*0201, DQA1*0501, DRB1*0301, TNFc2, TNFB*2, TNFal, TNFb5, B18. Thus, the IDDM‐associated TNF2 allele had no DR3‐independent value as a disease marker. The LPS‐induced TNF‐α production by human monocytes in relation to genotypes demonstrated that TNF1/2 heterozygous individuals had higher, though not statistically significantly (p = 0.08) levels than TNF1‐homozygous subjects. However, this difference was rather small, unlikely to be of biological significance and based on the present material we cannot establish the functional importance of this polymorphism.
Allergy‐associated Iϵ and Fcϵ receptor II (CD23b) genes activated via binding of an interleukin‐4‐induced transcription factor to a novel responsive element
Authors: Köhler Ingrid , Rieber Ernst Peter
Published: March 2007

Interleukin‐4 (IL‐4) has important regulatory functions in the immune system, particularly in the generation of immunoglobulin E, the principal mediator of allergic responses. The molecular basis of IL‐4 action has remained elusive so far. Here we report on a novel human transcription factor, termed nuclear factor IL‐4 (NF‐IL4), which is posttranslationally activated by IL‐4 in lymphoid and monocytic cells. Homologous binding sequences for NF‐IL4 were identified in the promoter regions of the IL‐4 controlled CD23b and Iϵ genes. We defined a palindromic 9‐bp consensus sequence (5′‐TYCYRRGAA‐3′) as IL‐4‐responsive element (IL‐4RE). Point mutation analysis of the CD23b promoter showed that binding of NF‐IL4 to the IL‐4RE is essential for the initiation of gene transcription in response to IL‐4. NF‐IL4 was not activated by Ca2+ ionophore, phorbol ester and cAMP either alone or in combination suggesting a non classical pathway for IL‐4 signal transduction.
Cyclosporin A inhibits T cell receptor‐induced interleukin‐2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme, protein kinase C‐β
Authors: Szamel Marta , Bartels F. , Resch K.
Published: March 2007

Stimulation of human peripheral blood lymphocytes via T cell receptor/CD3 complex resulted in a bimodal activation of protein kinase(s) C (PKC). Within 10 min of stimulation PKC‐α was translocated to, and thus activated in, the plasma membranes of human lymphocytes, followed by a fast dissociation of this isotype from the plasma membrane. This short term activation and translocation PKC‐α proved to be cyclosporin A (CsA) insensitive. After 90 min of stimulation PKC‐β was translocated to and remained bound to the plasma membranes for up to 4 h. Preincubation of human lymphocytes with 200 ng/ml CsA specifically and completely abolished the sustained activation of PKC‐β. Neither the phorbol ester‐induced direct activation of PKC nor the specific activity of the plasma membrane‐bound enzyme was influenced by CsA, suggesting that a signal transduction pathway leading to sustained activation of PKC‐β was influenced by the immunosuppressive agent. In fact, CsA inhibited, in a concentration‐dependent manner, the activation of lysophosphatid acyltransferase‐catalyzed elevated incorporation of cis‐polyunsaturated fatty acids into plasma membrane phospholipids. While interleukin‐2 (IL‐2) synthesis and cellular proliferation were completely inhibited by 200 ng/ml CsA in BMA 030‐ or BMA 031‐stimulated cells, expression of high‐affinity IL‐2 receptors was not influenced by the immunosuppressive drug. These results suggest that synthesis and expression of high‐affinity IL‐2 receptors might be regulated by a signal‐transducing pathway involving activation and translocation of PKC‐α. Lysophosphatid acyltransferase‐catalyzed incorporation of cis‐polyunsaturated fatty acids might represent another mechanism of signal transduction implicated in the activation and translocation of PKC‐β, which is specifically inhibited by CsA. Neutralization of PKC‐β by introducing anti‐PKC‐β antibodies prevented IL‐2 synthesis and proliferation in stimulated human lymphocytes. The results suggest a possible link between activation of PKC‐β and regulation of IL‐2 synthesis in activated human lymphocytes. Thus, inhibition of the activation and translocation of PKC‐β by CsA may result in inhibition of IL‐2 gene expression in human lymphocytes.
Differential effect of transporter Tap 2 gene introduction into RMA‐S cells on viral antigen processing
Authors: Ossevoort Miriam A. , Sijts Alice J. A. M. , van Veen Karin J. H. , Momburg Frank , Hämmerling Günter J. , Seelig Angela , Butcher Geoffrey W. , Howard Jonathan C. , Kast W. Martin , Melief Cornells J. M.
Published: March 2007

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing‐defective cell line RMA‐S (H‐2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA‐S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA‐S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus‐specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA‐S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H‐2d) or rat origin into RMA‐S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA‐S is not elevated by transfection of either Tap 2 gene into RMA‐S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA‐S on viral antigen processing.
The major histocompatibility complex influences myelin basic protein 63‐88‐induced T cell cytokine profile and experimental autoimmune encephalomyelitis
Authors: Mustafa Maha , Vingsbo Carina , Olsson Tomas , Ljungdahl Åke , Höjeberg Bo , Holmdahl Rikard
Published: March 2007

Polymorphism of the major histocompatibility complex (MHC) influences susceptibility to experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP) in rats. Current concepts relate such influences to the capacity of class II molecules to present relevant peptides to autoreactive T cells. We have here analyzed the MHC influence on the immune response and the development of EAE after immunization with the immunodominant peptide MBP‐63–88. Analysis of MHC‐congenic LEWIS strains showed that RT1a, RT1c and RT1l haplotypes are permissive for disease induction, whereas RT1d and RT1u are resistant. All EAE responding strains showed peptide‐specific proliferation and interferon (IFN)‐γ secretion, but no early significant tendency to express interleukin (IL‐4) or transforming growth factor (TGF)‐β mRNA in lymphocytes in response to the MBP 63–88, 7 days post immunization (p.i.). Later, 14 days p.i., peptide‐specific induction of IL‐4 and TGF‐β occurred in RT1l rats. Among the EAE non‐responders strains, only the RT1u rats showed an immune response to MBP 63‐88. This response, however, was qualitatively different from the immune response in the EAE‐susceptible strains. Thus, there was no proliferation and only moderate IFN‐γ production in response to peptide, but in contrast, a significant and early peptide‐induced IL‐4 and TGF‐β response was observed. The data suggest that the MHC‐associated susceptibility to EAE is partly related to the ability to mount a TH1‐like immune response while the MHC‐associated EAE resistance may either be related to MBP peptide non‐responsiveness or to peptide recognition and induction of a qualitatively different and disease down‐regulatory immune response.
The Bruton's tyrosine kinase gene is expressed throughout B cell differentiation, from early precursor B cell stages preceding immunoglobulin gene rearrangement up to mature B cell stages
Authors: de Weers Michel , Verschuren Martie C. M. , Kraakman Margriet E. M. , Mensink Rob G. J. , Schuurman Ruud K. B. , van Dongen Jacques J. M. , Hendriks Rudolf W.
Published: March 2007

X‐linked agammaglobulinemia (XLA) is an immunodeficiency disease in man, resulting from an arrest in early B cell differentiation. The gene defective in XLA has recently been identified and encodes a cytoplasmic protein tyrosine kinase, named Bruton's tyrosine kinase (btk), essential for cell differentiation and proliferation at the transition from pre‐B to later B cell stages. In this study we investigated btk expression by Northern blotting experiments in a series of human (precursor‐) B cell lines, acute lymphoblastic leukemias and plasmacytomas, btk was found to be already expressed in very early stages of B cell differentiation, even prior to immunoglobulin (Ig) heavy (H) or light (L) chain gene rearrangements. Transcripts were also detected at the pre‐B cell stage and in mature B cells, irrespective of the Ig H chain class expressed. Approximately at the transition from mature B cells to plasma cells, expression of the btk gene is down‐regulated. In addition, the btk gene was found to be expressed in myeloid cell lines and acute myeloid leukemias. btk expression in myeloid cells is probably not a prerequisite for myeloid differentiation, since myeloid cells in XL A patients seem not to be affected. No btk expression was found in T‐lineage cells. The btk expression profile, i.e. from early precursor‐B cell stages preceding Ig rearrangement up to mature B cells, supports the hypothesis that the XLA defect resides in a critical step of B cell development which is independent of the Ig gene recombination machinery.
Increased number of cytotoxic T cells within CD4+8− T cells in β2‐microglobulin, major histocompatibility complex class I‐deficient mice
Authors: Marušić‐Galešić Suzana , Udaka Keiko , Walden Peter
Published: March 2007

Targeted disruption of β2‐microgobulin gene results in deficient major histocompatibility complex class I expression and failure to develop CD4−8+ T cells. Despite this, β2M−/− mice reject skin grafts and cope with most viral infections tested. We asked whether CD4+8− cytotoxic T cells could play a role in compensating for the defect in CD4−8+ cytotoxic T cell function. We found that the cytotoxic activity against class II+ targets is significantly higher among CD4+8− T cells of β2M−/− than among those of β2M+/+ mice. In the limiting dilution experiment, we showed that the precursor frequency for the cytotoxic, CD4+8−, class II‐specific T cells is at least fivefold higher in β2M −/−than in β2M+/+ mice. These results suggest that CD4+8− cytotoxic T cells could play a major role in carrying out cytotoxic function in β2M−/− mice.
Phenotypical and functional characterization of Fey receptor I (CD64)‐negative monocytes, a minor human monocyte subpopulation with high accessory and antiviral activity
Authors: Grage‐Griebenow Evelin , Lorenzen Dirk , Fetting Rudolf , Flad Hans‐Dieter , Ernst Martin
Published: March 2007

Fcγ receptor I‐positive (CD64+) and Fcγ receptor I‐negative (CD64−) monocytes were prepared from highly purified (elutriation‐derived) human monocytes by cytofluorograph cell sorting, and a phenotypical and functional dissociation of the isolated CD64+ and CD64− monocyte subsets is demonstrated. Surface analyses revealed that the surface antigen pattern of CD64+ monocytes corresponds to the phenotype of typical unseparated monocytes. In contrast, CD64− monocytes are characterized by high expression of major histocompatibility complex (MHC) class I antigens (HLA‐A, ‐B, ‐C) and MHC class II antigens (HLA‐DR, ‐DP, ‐DQ), and low expression of the monocyte‐specific marker CD14 which is found on nearly all CD64+ monocytes. However, 75% of the CD64− cells were found to be esterase‐positive, and 85% were positive for the monocyte/macrophage‐specific intracellular antigen CD68. Furthermore, CD64+ monocytes show significantly higher expression of CD45RA and Fcγ receptor III (CD16) than CD64+ monocytes, but lack the natural killer cell markers CD56 and CD57. Functional studies showed that cells of the minor CD64− monocyte subset have a higher accessory cell capacity in antigen‐driven T cell activation than CD64+ monocytes. CD64− monocytes pretreated with PPD (purified protein derivative of tuberculin) induced up to tentimes more interferon‐γ and also higher proliferation in responding autologous T cells than PPD‐pretreated CD64+ monocytes. Similar results were obtained for T cells in mixed leukocyte reaction. Interferon‐γ release and proliferation of allogeneic lymphocytes were consistently higher in the presence of irradiated CD64− monocytes than of irradiated CD64+ monocytes. Furthermore, when CD64− and CD64+ monocytes were stimulated with Newcastle disease virus, we measured an up to 67‐fold higher interferon‐a release from CD64− than from CD64+ monocytes, indicating a higher anti‐viral capacity of this subset. CD64− monocytes showed lower activity in the phagocytosis of unopsonized particles and also lower zymosan‐ or latex‐induced chemiluminescence than CD64+ monocytes. These findings indicate that CD64− monocytes, although comprising only less than 10% of all peripheral blood monocytes, represent a monocyte subpopulation efficiently interacting in vitro with T cells and, additionally, are the major source of interferon‐α.
Antigen‐independent adhesion of CD4 CD45RA T cells from cord blood
Authors: Buzyn‐Veil Agnès , Hivroz Claire , Lecomte Olivia , Barbat Christiane , Mazerolles Fabienne , Fischer Alain
Published: March 2007

Antigen‐independent adhesion of resting adult CD4+ CD45RO+ T cells to B lymphocytes has been shown to be transient and can be down‐regulated by CD4 major histocompatibility complex (MHC) class II molecule interactions. Conversely, adhesion of adult CD4+ CD45RA+ subpopulation to B cells is not regulated by ligands of CD4. We have investigated the regulation of adhesion of cord blood CD45RA+ CD4+ T lymphocytes. In contrast to adult CD45RA+ CD4+ T cells, cord blood CD45RA+ CD4+ T cells were strongly sensitive to the down‐regulation of adhesion mediated by the CD4‐HLA class II interaction, since adhesion to MHC class II(+) B cells was transient and inhibited by an anti‐CD4 antibody. In addition, human immunodeficiency virus gpl60, synthetic gpl06‐derived peptides encompassing a CD4 binding site inhibited conjugate formation between cord blood CD45RA+ CD4+ T cells and B cells. Following activation of the cord blood CD4 T cells by an anti‐CD3 antibody, a conversion from a transient to a stable adhesion pattern of cord blood CD4 T cells to B cells occurred in 2 days. The reversal to a transient adhesion occurred at day 8 following anti‐CD3 activation in correlation with a complete shift to a CD45RO phenotype of the cord blood CD4 T cells. These data suggest that CD4 T cell adhesion can be developmentally regulated.
Development of T cells in SCID mice grafted with fetal thymus from AKR mice or F344 rats
Authors: Maeda Ken‐Ichi , Nagasawa Hideyuki , Furukawa Atsuko , Hisaeda Hajime , Maekawa Yoichi , Manabe Tetsuya , Kudo Eiji , Good Robert A. , Himeno Kunisuke
Published: March 2007

To examine the development of T cells within an allogeneic or xenogeneic environment, we engrafted the fetal thymus from AKR mice or F344 rats under the kidney capsule of SCID mice (mTG and rTG mice). T lymphopoiesis developed in SCID mice 2 months after transplantation, although the ratio of CD4/CD8 in both experimental groups was different from that of normal control. T cells in mTG mice did not show in vitro proliferation or cytotoxicity against either host‐type C.B‐17 (H‐2d) or donor‐type AKR (H‐2k) cells, while they exerted potent activities against third‐party BIO (H‐2b) cells. In contrast, T cells in rTG mice exhibited proliferation against both host‐type C.B‐17 and donor‐type F344 rat cells. Consistently, graft‐vs.‐host disease symptoms developed in these mice and histological examination showed impressive infiltration of lymphocytes into the skin or into the mucosal layers of the stomach. Activated state of T cells in rTG mice was also evidenced by the positive expression of interleukin‐2 receptor. Taken together, fetal thymus appears to contain progenitor cells which are sufficient for in vivo reconstitution of T lymphopoiesis, but species‐specific environment is important for the induction of tolerance. In mTG mice, Vβ6+ T cells reactive to donor Mlsa determinants and Vβ3+ T cells reactive to host Mlsc determinants were deleted, suggesting that tolerance was regulated mainly by clonal deletion. By contrast, Vβ11+ T cells reactive to Mlsf determinants were not deleted possibly due to the lack of their ligands.
The invariant chain induces compact forms of class II molecules localized in late endosomal compartments
Authors: Humbert Martine , Raposo Graça , Cosson Pierre , Reggio Hubert , Davoust Jean , Salamero Jean
Published: March 2007

The invariant chain (Ii) binds to newly synthesized major histocompatibility complex (MHC) class II molecules and is targeted to an acidic compartment where it is degraded. To evaluate its role on the conformation and the subcellular distribution of murine MHC class II molecules we have established stable L cell transfectants expressing class II IAk heterodimers alone or in conjunction with p31 and p41 Ii chains. In these cells, class II molecules were present under three forms: αβ heterodimers bearing high mannose carbohydrate moieties, and fully glycosylated αβ heterodimers that are sensitive or resistant to sodium dodecyl sulfate dissociation at 20 °C. The latter class II molecules called compact heterodimers, were here highly induced in Ii‐positive cells. Using in situ iodination of endosomal compartments, class II heterodimers were detected in late endosomal compartments essentially as compact forms in Ii‐positive cells, and as non‐compact forms in Ii‐negative cells. Using confocal microscopy, IAk molecules were located in compartments distinct from early endosomes labeled with transferrin, but partially coincident with vesicles containing fluid‐phase markers, and highly coincident with compartments containing large amounts of cathepsins B, D, H, and L in Ii‐positive and Ii‐negative cells. At the ultrastructural level, class II molecules were mostly present in multivesicular bodies, even without Ii expression. But Ii chains were needed to induce an efficient presentation of the hen egg lysozyme antigen and were sufficient to promote a major conformational change of the late endosomal, and/or lysosomal resident, class II molecules. Ii molecules are presumably playing a chaperoning function favoring the association of peptides with class II molecules in endosomal compartments.
Generation of a novel CD8+ cytotoxic T lymphocyte that requires soluble factor to lyse autologous antigen‐presenting cells
Authors: Yoshizumi Hideyuki , Kamikawaji Nobuhiro , Nishimura Yasuharu , Sasazuki Takehiko
Published: March 2007

We reported the existence of high and low responders to the streptococcal cell wall antigen (SCW) in the human population. To analyze the mechanism of the low responsiveness to SCW at the cellular level, we established SCW‐specific CD4+ T cell lines. During the course of generation of a SCW‐specific CD4+ T cell line restricted by HLA‐DQ from a low responder, we obtained autoreactive CD8+ cytotoxic T lymphocytes as a cell line (HYCD8). They proliferated in the presence of autologous monocytes and IL‐2, without SCW. HYCD8 lysed autologous monocytes and Epstein‐Barr virus‐transformed B lymphoblastoid cell line (BLCL). This cytotoxic activity was specifically inhibited by an anti‐HLA class I framework monoclonal antibody and restricted by HLA‐B52 or B54 specificity, as judged by killing activity against panel cells and HLA class I‐transfected BLCL. It was unique to HYCD8 that the HLA class I‐restricted cytotoxicity was observed only in the presence of soluble factor with low molecular mass (< 104 Da) produced mainly by B cells, which could not be replaced by known cytokines and their mixtures. We thus describe novel HLA class I‐restricted cytotoxic CD8+ T cells that kill antigen‐presenting cells in a soluble factor‐dependent manner.
Identification of new B cell epitopes in the sera of rheumatoid arthritis patients using a random nanopeptide phage library
Authors: Dybwad Anne , Førre Øystein , Kjeldsen‐Kragh Jens , Natvig Jacob B. , Sioud Mouldy
Published: March 2007

A random nanopeptide phage library was used to screen a pool of immunoglobulin fractions obtained from rheumatoid arthritis (RA) patients. After three rounds of panning, random individual phages were selected by their capacity to react with individual sera from RA patients.

By sequencing the inserts corresponding to the peptides displayed on the surface of the phages, we found that phages displaying particular peptides were overrepresented in the selected libraries. The peptides displayed by these phages were: pep1 = Ala‐Asp‐Gly‐Gly‐Ala‐Gln‐Gly‐Thr‐Ala; pep2 = Pro‐Gly‐Pro‐Ser‐Arg‐Ala‐His‐Phe‐Leu; pep3 = Leu‐Ser‐Ser‐Arg‐Glu‐Pro‐Gln‐Ala‐Arg; pep4 = Arg‐Leu‐Thr‐Arg‐Glu‐Leu‐Tyr‐Ala‐Gln and pep5 = Tyr‐Thr‐Gln‐Lys‐His‐Gln‐Ala.

The percentage of sera positive for pep1 was higher in RA patients as compared to the normal adults (p 0.0004) and the reacting antibody was mainly of IgG isotype. The specificity of binding to the phage displaying pep1 was confirmed by competition experiments using both isolated phages and a synthetic peptide. Interestingly, a mutated phage displaying only Ala‐Asp‐Gln‐Gly‐Thr‐Ala had no significant reactivity with the sera, indicating that the amino acids (Gly‐Gly‐Ala) of pep1 are the vital for the binding. Taken together this study demonstrates that it is possible to select specific ligands from a random phage library using sera from RA patients. In addition, this approach could be useful for identifying peptide antigens that might be part of causitive agents in autoimmune diseases.
Carrier‐mediated uptake and presentation of a major histocompatibility complex class I‐restricted peptide
Authors: Brander Christian , Wyss‐Coray Tony , Mauri Davide , Bettens Florence , Pichler Werner J.
Published: March 2007

Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I‐restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA‐A2‐restricted influenza virus matrix protein‐derived peptide (flu, 57–68) was used either in soluble form or coupled via an S‐S bridge to transferrin (Tf‐flu). Target cells were incubated with flu or Tf‐flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide‐specific, HLA‐A2‐restricted CD8+ cytotoxic T lymphocytes. Sensitization of target cells with Tf‐flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf‐flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf‐flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf‐flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1‐buthionine‐(s, r)‐sulfoximine or n‐ethylmaleimide. Presentation of the flu peptide derived from Tf‐flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP‐independent steps.
Major histocompatibility complex class I‐binding peptides are recycled to the cell surface after internalization
Authors: Abdel Motal Ussama M. , Zhou Xianzheng , Joki Annalena , Siddiqi Abdur Rehman , Srinivasa B. R. , Stenvall Kristina , Dahmén Jan , Jondal Mikael
Published: March 2007

Cytotoxic T lymphocytes (CTL) recognize target antigens as short, processed peptides bound to major histocompatibility complex class I (MHC‐I) heavy and light chains (β2‐microglobuhn; β2 m).The heavy chain, which comprise the actual peptide binding α‐1 and α‐2 domains, can exist at the cell surface in different forms, either free, bound to β2m or as a ternary complex with β2m and peptides. MHC‐I chains are also known to internalize, and recycle to the cell surface, and this has been suggested to be important in peptide presentation. Whether MHC‐I‐bound peptides also can recycle is not known. We have investigated this by using both peptide transporter mutant RMA‐S cells and EL4 cells loaded with Db‐binding peptides, by two different approaches. First, peptides were covalently linked with galabiose (Galα4Gal) at a position which did not interfere with Db binding or immunogenicity, and peptide recycling tested with Gal2‐specific monoclonal antibodies. By flow cytometry, a return of Gal2 epitopes to the cell surface was found, after cellular internalization and cell surface clearance by pronase treatment. This peptide recycling could be discriminated from free fluid‐phase uptake and was inhibited by methylamine, chloroquine and low temperature (18°C) but not by leupeptin. Second, specific CTL were reacted with peptide‐loaded target cells after complete removal of surface Db molecules by pronase, and after different times of incubation at 37C to allow reexpression. By this procedure, reappearance of target cell susceptibility was confirmed. The results are in agreement with a model for optimizing peptide presentation by recycling through an intracellular compartment similar to early endosomes in certain antigen‐presenting cells.
IgE‐binding molecules (Mac‐2/ϵBP) expressed by human eosinophils. Implication in IgE‐dependent eosinophil cytotoxicity
Authors: Truong Marie‐José , Gruart Valérie , Liu Fu‐Tong , Prin Lionel , Capron André , Capron Monique
Published: March 2007

Macrophage cell‐surface protein 2 (Mac‐2), a galactose specific S‐type lectin identified in inflammatory macrophages, presents a high degree of homology with the rat IgE‐binding protein (ϵBP). In the present study, we show by different experimental approaches that human eosinophils can express Mac‐2/ϵBP. Flow cytometry analysis revealed that a large proportion of eosinophilic patients expressing binding sites for IgE on their eosinophil membrane, were able to bind anti‐Mac‐2 monoclonal antibody (mAb). Northern blot performed with eosinophil RNA hybridized with the human Mac‐2 or ϵBP cDNA probes revealed that eosinophils presented a unique transcript at 1.2 kb. Immunoprecipitation of eosinophil extracts with anti‐Mac‐2 mAb revealed the presence of a molecule of 29 kDa corresponding to Mac‐2 protein, as well as one additional molecule of 15 kDa, absent from control alveolar macrophages. The function of these molecules was investigated in a radiolabeled IgE binding assay. Anti‐Mac‐2 mAb as well as galactose and lactose saccharides significantly inhibited the binding of radiolabeled human myeloma IgE protein to eosinophils. Moreover, the dose‐dependent inhibition by anti‐Mac‐2 mAb of IgE‐dependent eosinophil‐mediated cytotoxicity towards parasite targets indicated the role of these IgE‐binding molecules in the function of human eosinophils. These results suggest that in addition to transmembrane receptors, lectin‐type molecules can participate in the IgE‐dependent effector function of eosinophils.
Enhancement of disease by neutralizing antiviral antibodies in the absence of primed antiviral cytotoxic T cells
Authors: Battegay Manuel , Kyburz Diego , Hengartner Hans , Zinkernagel Rolf M.
Published: March 2007

The effects of neutralizing antibodies on the disease course in mice infected with the noncytopathic lymphocytic choriomeningitis virus (LCMV) were evaluated. Whereas non‐neutralizing antisera exhibiting high enzyme ‐ linked immunosorbent assay titers had no effect on T cell responses and their consequences, neutralizing antisera modulated them variably. Neutralizing antibodies were able to prevent lethal choriomeningitis after intracerebral infection with a neurotropic LCMV‐isolate (ARMSTRONG) although they could not control local virus replication. The same antibodies exhibited little or no protective effect on choriomeningitis induced by LCMV‐WE, a viscerotrope isolate. Surprisingly, these antibodies rendered mice much more susceptible to choriomeningitis after intracerebral infection with LCMV DOCILE, a very rapidly spreading lymphocyto‐viscerotrope virus; in this situation antibodies prevented overwhelming infection which causes deletion of immunopathogenic cytotoxic T cell responses. Thus preexisting neutralizing antiviral antibodies had little influence on local virus spread in peripheral tissues but they reduced hematogenic spread and infection of antigen‐presenting cells; thereby they influenced the primary cytotoxic T cell (CTL) response and indirectly modulated the extent of T cell‐mediated immunopathology in peripheral organs. These results may explain why vaccines inducing neutralizing antibodies but no CTL may enhance an immunopathological disease caused by challenge infection with a noncytopathic virus.
Human IgM+IgD+ B cells, the major B cell subset in the peripheral blood, express Vϰ genes with no or little somatic mutation throughout life
Authors: Klein Ulf , Küppers Ralf , Rajewsky Klaus
Published: March 2007

Peripheral blood B cells of a 67‐year‐old person were separated into IgM+IgD+, IgM+IgD−, and IgM−IgD− subsets, and nucleotide sequences of expressed immunoglobulin light chain variable (V) regions encoded by Vϰ3 and Vϰ4 gene family members were determined from amplified cDNA. V region sequences from IgM+IgD+ cells (the major B cell population in the blood) showed no or little somatic mutation (0.3%), in contrast to Vϰ sequences from IgM+IgD− and IgM−IgD− B cells (2.0% and 3.9%, respectively). This suggests that in the human like in the mouse, and independently of age, somatically mutated memory B cells accumulate in the compartment of IgM−IgD− cells, whereas the IgM+IgD+ subpopulation consists of cells whose antibody repertoire is mainly determined by V region gene rearrangements and N‐region insertion, at the molecular level. The somatically mutated IgM+IgD− cells may represent early descendants of IgM+IgD+ cells recruited into the memory cell compartment.
Activation of MAP kinases, pp90rsk and pp70‐S6 kinases in mouse mast cells by signaling through the c‐kit receptor tyrosine kinase or FcϵRI: rapamycin inhibits activation of pp70‐S6 kinase and proliferation in mouse mast cells
Authors: Tsai Mindy , Chen Rey‐Huei , Tam See‐Ying , Blenis John , Galli Stephen J.
Published: March 2007

The high‐affinity receptor for IgE, FcϵRI, represents the major cell surface structure through which mast cells express immunologically specific secretory function. By contrast, the stem cell factor receptor (SCFR), which is encoded by c‐kit, is essential for normal mast cell development. The signaling pathways initiated by the stimulation of mast cells through the FcϵRI, which lacks intrinsic kinase activity, and the SCFR, a member of the receptor tyrosine kinase family, generally have been regarded to be distinct. We report here that mouse mast cells stimulated either with SCF or with IgE and specific antigen exhibit a remarkably similar pattern of activation of mitogen‐activated protein kinases (MAPK), 90 kDa‐S6 kinases (pp90rsk), and pp70‐S6 kinases (pp70‐S6K). These results indicate that all three families of protein kinases are associated with the cell surface receptor‐dependent activation of secretion, as well as proliferation, in mast cells. We also show that the immunosuppressant rapamycin, but not FK506, can inhibit both SCF‐dependent pp70‐S6 kinase activation and SCF‐dependent proliferation in mouse mast cells, without suppressing IgE‐ and antigen‐dependent mediator release. These findings suggest that the activation of pp70‐S6 kinase represents an important link in the stimulation of cell proliferation by SCF. Our results also indicate that the intracellular signaling pathways initiated by stimulation of mast cells through the FcϵRI or the SCFR exhibit more overlap than has previously been appreciated.
Role of LFA‐1/ICAM‐1 in interleukin‐2‐stimulated lymphocyte proliferation
Authors: Vyth‐Dreese Florry A. , Dellemijn Trees A. M. , Frijhoff Anita , van Kooyk Yvette , Figdor Carl G.
Published: March 2007

Major adhesion routes between lymphoid cells involve the receptor/ligand pairs LFA‐l/ICAM‐1 and CD2/LFA‐3, in addition to VLA or CD44 molecules. In this study we evaluated the role of these adhesion receptors in the proliferative response of lymphoid cells to interleukin‐2 (IL‐2). Blocking studies were performed with a panel of monoclonal antibodies (mAb) directed against these adhesion molecules. Selective inhibition of recombinant (r)IL‐2‐induced cell proliferation was observed with mAb directed against the a or /3 subunit of LFA‐1 or to its ligand ICAM‐1. Interestingly, rIL‐2‐induced proliferation was also inhibited by NKI‐L16, an anti‐la antibody known to enhance cell‐cell interaction. Resting lymphocytes were preferentially susceptible to the inhibition, particularly in an early phase of culture and when stimulated with a relatively low dose of rIL‐2. By using mAb that specifically could block distinct rIL‐2 activation pathways, LFA‐l/ICAM‐1 interaction was found to be required for p55 IL‐2 receptor (IL‐2R)‐mediated interaction of rIL‐2 with its high‐affinity receptor, but not for p75 IL‐2R‐mediated responses. Furthermore, it was shown that the rIL‐2 response of T lymphocytes, but not of natural killer cells, was dependent on LFA‐l/ICAM‐1 interaction. This suggests that LFA‐l/ICAM‐1 interaction is required for an optimal rIL‐2 response of cells capable of IL‐2 secretion. Our data provide evidence for the hypothesis that adhesion receptor‐directed release of IL‐2 may result in a locally high concentration of IL‐2 that triggers high‐affinity IL‐2R signaling and up‐regulates p55 IL‐2R to enhance cytokine responsiveness.