bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockade of CD28/B7 co‐stimulation by mCTLA4‐Hγ1 inhibits antigen‐induced lung eosinophilia but not Th2 cell development or recruitment in the lung
Authors: Harris Nicola , Campbell Chloë , Le Gros Graham , Ronchese Franca
Published: March 2007

We have studied the role of the CD28/B7 co‐stimulatory pathway in the development of a Th2‐type lung immune response. Mice injected two or three times intraperitoneally with ovalbumin in alum adjuvant and then re‐exposed to the same antigen by intranasal (i.n.) inoculation show infiltration of the lung tissue and appearance in the broncho‐alveolar lavage (BAL) fluid of significant numbers of eosinophils and lymphocytes, in a pattern which is reminiscent of asthmatic inflammation. The accumulation of eosinophils in the airways is completely dependent on interleukin (IL)‐5 secretion by CD4+ T cells. We have used mice transgenic for a soluble form of murine CTLA‐4 (mCTLA4‐Hγ1) which binds to B7 molecules on antigen‐presenting cells, thereby preventing their interaction with T cell‐expressed CD28. mCTLA4‐Hγ1‐transgenic mice immunized intraperitoneally and challenged i.n. with ovalbumin failed to generate any eosinophil infiltration, suggesting that little or no IL‐5 was secreted in the lungs of these mice. In contrast with the complete lack of eosinophils, the numbers and phenotypes of infiltrating lymphocytes were comparable in the lungs of mCTLA4‐Hγ1‐transgenic and normal mice. Also, lung lymphocytes from immunized mCTLA4‐Hγ1‐transgenic and normal mice could be shown to secrete comparable amounts of IL‐4 and IL‐5 when stimulated in culture in the absence of mCTLA4‐Hγ1. We conclude that mCTLA4‐Hγ1 can efficiently block the production of IL‐5 during in vivo responses and inhibit eosinophil recruitment, but that it does not block the development of CD4+ T cells into Th2 cells with the potential to secrete IL‐5.
Human naive B cells cultured with EL‐4 T cells mimic a germinal center‐related B cell stage before generating plasma cells. Concordant changes in Bcl‐2 protein and messenger RNA levels
Authors: Grimaǐtre Michel , Werner‐Favre Christiane , Kindler Vincent , Zubler Rudolf H.
Published: March 2007

The T cell‐dependent B cell response in vivo occurs in organized microenvironments. Alternative routes exist in that early plasma cells are generated in the T zone white others emerge later from the germinal center (GC) reaction. We investigated whether B cell stages resembling those defined in vivolex vivo might be induced in an in vitro system in which naive human B cells are activated by EL‐4 T cells and cytokines. Adult peripheral blood‐ or cord blood‐derived B cells were found to mimic an early activated stage (CD38low, IgD+, increased CD5+) followed by a centroblastic GC‐related stage (CD38int, CD77+, CD95(Fas)+, Bcl‐2 proteinlow) before differentiating into morphologically typical, CD38high, Fas‐ plasma cells of an immature type (Bcl‐2low, VLA‐5‐). The GC‐related cells and the plasma cells exhibited spontaneous apoptosis in medium, the former also undergoing anti‐Fas antibody‐induced apoptosis in medium as well as during CD40L exposure in the EL‐4 cultures. These Bcl‐2low cells maintained a high viability in contact with EL‐4 cells. Thus, some, major B cell stages with typical functional features as described for cells in vivolex vivo are sequentially generated in this in vitro system and the kinetics of the changes can be analyzed in a synchronized cell population. With regard to previous apparently conflicting observations on the Bcl‐2 mRNA level in GC B cells, we performed competitive reverse‐transcription polymerase chain reaction. Concordant changes in Bcl‐2 mRNA and protein levels were found, i.e. during Bcl‐2 down‐regulation in the GC‐related B cells in ongoing EL‐4 cultures or in medium, and during a more modest up‐regulation upon contact with fresh EL‐4 cells. Regulation of Bcl‐2 protein, therefore, predominantly occurred at the mRNA steady‐state level.
Prevention of autoimmune diabetes mellitus in NOD mice by transgenic expression of soluble tumor necrosis factor receptor p55
Authors: Hunger Robert E. , Carnaud Claude , Garcia Irène , Vassalli Pierre , Mueller Christoph
Published: March 2007

The non‐obese diabetic (NOD) mouse represents a relevant animal model of autoimmunity for insulin‐dependent diabetes mellitus. The pathogenic role of tumor necrosis factor (TNF) in insulitis and β cell destruction observed in these mice remains controversial, since injections of TNF or of anti‐TNF antibodies have been reported to exert protection or acceleration of diabetes, depending on the timing of administration. In this study, we demonstrate that, in contrast to the non‐transgenic littermates, NOD mice with permanent neutralization of TNF by high blood levels of soluble TNF receptor p55‐human FcIgG3‐fusion molecules resulting from the expression of a transgene are protected from spontaneous diabetes. They are also protected from accelerated forms of disease caused by transfer of NOD spleen cells or cyclophosphamide injections. This protection is associated with a marked decrease in the severity and incidence of insulitis and in the expression of the adhesion molecules MAdCAM‐1 and ICAM‐1 on the venules of pancreatic islets. These data suggest a central role for TNF‐α in the mediation of insulitis and of the subsequent destruction of insulin‐secreting β‐cells observed in NOD mice. They may be relevant to cell‐mediated autoimmune diseases in general, in which treatment with soluble TNF receptors might be beneficial.
Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fcε receptors
Authors: Amoui Mehran , Dráberová Lubica , Tolar Pavel , Dráber Petr
Published: March 2007

Activation of rat mast cells through the receptor with high affinity for IgE (FcεRI) requires a complex set of interactions involving transmembrane subunits of the FcεRI and two classes of nonreceptor protein tyrosine kinase (PTK), the Src family PTK p53/p56lyn (Lyn) and the Syk/ZAP‐family PTK p72syk (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated FcεRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of FcεRI subunits in this process, we have analyzed Syk‐ and Lyn‐associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of FcεRI β or γ subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet‐P40‐solubilized antigen (Ag)‐ or pervanadate‐activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag‐ or pervanadate‐activated cells were used. A similar amount of Syk was bound when lysates from pervanadate‐activated variant cells deficient in the expression of FcεRI β or γ subunits were used, suggesting that FcεRI does not function as the only intermediate in the formation of the Syk‐Lyn complexes. Further experiments have indicated that Syk‐Lyn interactions occur in Ag‐activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast‐like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in FcεRI signaling.
Lack of F1 anti‐parental resistance in H‐2b/d F1 hybrids devoid of β2‐microglobulin
Authors: Höglund Petter , Olsson‐Alheim Mats Y. , Johansson Maria , Kärre Klas , Sentman Charles L.
Published: March 2007

F1 hybrid mice often reject parental hematopoietic grafts, a phenomenon known as hybrid resistance. Hybrid resistance is mediated by natural killer (NK) cells and although the molecular interactions responsible for this phenomenon are largely unknown, one hypothesis suggests that parental cells are rejected because they fail to express a complete set of host major histocompatibility complex (MHC) class I molecules. Inherent in this theory is that NK cells in the F1 hybrid are instructed by self MHC class I molecules to form an NK cell repertoire capable of reacting against cells lacking these self MHC class I molecules. Here, we show that C57BL/6 x DBA/2 mice (H‐2b/d) devoid of β2‐microglobulin (β2m) are incapable of rejecting β2m−/− parental C57BL/6 cells (H‐2b) both in vivo and in vitro. From this, we conclude that the development of an NK cell repertoire, at least in F1 mice of the H‐2b/d haplotype, requires expression of MHC class I molecules complexed with β2m.
Susceptibility to ankylosing spondylitis correlates with the C‐terminal residue of peptides presented by various HLA‐B27 subtypes
Authors: Fiorillo Maria Teresa , Meadows Leslie , D'Amato Mauro , Shabanowitz Jeffrey , Hunt Donald F. , Appella Ettore , Sorrentino Rosa
Published: March 2007

Susceptibility to spondyloarthropaties is strongly associated with some HLA‐B27 alleles. Evidence suggests a direct pathogenic role for the B27 molecules which possibly present an arthritogenic peptide to the T cells. If this hypothesis is true, B27 subtypes that differ structurally but are disease‐associated ought to be capable of presenting such peptide(s), while non‐disease‐associated ones would not. We have recently described a B27 subtype, B*2709, and shown its absence in ankylosing spondylitis (AS) patients. Here, we show the elution and sequence of peptides from HLA‐B*2709 molecules. Similar to other B27 subtypes, these peptides are mainly nonamers with an Arg at position P2. Comparison of the C‐terminal anchors of peptides eluted from B*2702 and B*2705 with those eluted from B*2709 reveals that, while B*2702 and B*2705 have a broader specificity, B*2709 molecules appear to only accept C‐terminal hydrophobic residues. A common feature shared by the two caucasoid AS‐associated subtypes (B*2702 and B*2705) but different from B*2709, is the presence of a Tyr as peptide C‐terminal anchor. The substitution of Val for Tyr at the C terminus in one of the eluted peptides greatly reduces the binding to B*2709 molecules. This finding suggests Tyr as a discriminative amino acid allowed at the C terminus of peptides bound to the AS‐associated B27 subtypes, but not to those which are not associated with AS.
CD45‐associated protein is a lymphocyte‐specific membrane protein expressed in two distinct forms
Authors: Kitamura Koichi , Matsuda Akio , Motoya Satoshi , Takeda Akiko
Published: March 2007

CD45‐AP is a recently identified CD45‐associated protein. The two proteins interact specifically through their respective transmembrane segments. Northern hybridization analysis of CD45+ T lymphocytes and their CD45− variants demonstrated that the production of CD45‐AP and CD45 mRNA is regulated independently. On the other hand, Western blotting analysis indicated that the CD45‐AP protein has a shorter half‐life in the absence of CD45 in three out of four variants. Similar analysis of various types of leukocytes demonstrated that CD45‐AP is expressed in T, B, and pre‐B cells, but not in plasma cells or cells of the monocyte/macrophage lineage. Two forms of CD45‐AP mRNA exist in all types of CD45‐AP‐expressing lymphocytes analyzed. One form corresponds to the previously reported CD45‐AP cDNA and the other form encodes an additional 12 amino acids at the N terminus. The two CD45‐AP proteins are identical in their capacity for specific binding to CD45, but employ different mechanisms for endoplasmic reticulum membrane translocation.
gp160 of HIV or anti‐CD4 monoclonal antibody ligation of CD4 induces inhibition of JNK and ERK‐2 activities in human peripheral CD4+ T lymphocytes
Authors: Jabado Nada , Pallier Annaïck , Jauliac Sébastien , Fischer Alain , Hivroz Claire
Published: March 2007

Under physiological conditions, activation of CD4+ T cells by major histocom‐patibility complex (MHC) antigen complexes requires engagement of both the T cell receptor and the CD4 molecule. However, CD4 ligands binding to the CD4 molecule has also been shown to inhibit T cell proliferation and interleukin (IL)‐2 production in human CD4+ T cells, in an MHC‐independent way. We have previously shown that this inhibition was associated with a diminished binding activity of the IL‐2 transcription factors NF‐AT, NF‐χB, and AP‐1. AP‐1 plays a key role in the regulation of IL‐2 transcription, and ERK and JNK activities are necessary for regulating AP‐1 at both the transcriptional and the post‐transcriptional levels. We therefore studied, in human peripheral CD4+ T cells, the regulation of the activities of extracellular signal‐regulated protein kinases (ERK) and c‐Jun N‐terminal kinases (JNK) by two CD4 ligands, gpi60 the envelope glycoprotein of human immunodeficiency virus (HIV) and an anti‐CD4 monoclonal antibody (mAb). Pre‐incubation of CD4+ T lymphocytes in the presence of anti‐CD4 mAb or gp160 inhibits the activation of JNK in response to phorbol 12‐myristate 13‐acetate and ionomycin. In the same conditions, phos‐phorylation and activation of ERK‐2 were also inhibited. Inhibition of both JNK and ERK‐2 activities are specific for binding of CD4 ligands to the CD4 molecule. They were not observed in CD8+ T lymphocytes. These results suggest that a specific inhibition of JNK and ERK‐2 activities contributes to defective IL‐2 production in T lymphocytes pre‐incubated with CD4 ligands.
On the mechanisms of immunodominance in cytotoxic T lymphocyte responses to minor histocompatibility antigens
Authors: Pion Stéphane , Fontaine Pierre , Desaulniers Michel , Jutras Julie , Filep Janos G. , Perreault Claude
Published: March 2007

Although there are numerous minor histocompatibility antigens (MiHA), T cell responses leading to graft‐versus‐host (GVH) and graft‐versus‐tumor effects involve only a small number of immunodominant MiHA. The goal of the present study was to analyze at the cellular and molecular levels the mechanisms responsible for MiHA immunodominance. Cytotoxic T lymphocytes (CTL) generated in eight combinations of H2b strains of mice were tested against syngeneic targets sensitized with HPLC‐fractionated peptides eluted from immunizing cells. The number of dominant MiHA was found to range from as little as two up to ten depending on the strain combination used. The nature of dominant MiHA was influenced by both the antigen profile of the antigen‐presenting cells (APC) and the repertoire of responding CTL. When C57BL/6 dominant MiHA (B6dom) and H‐Y were presented on separate APC, they showed similar immunogenicity. In contrast, when they were presented on the same APC, B6dom MiHA totally dominated H‐Y. B6dom MiHA did not suppress anti‐H‐Y responses by acting as T cell receptor antagonists for anti‐H‐Y CTL, nor were anti‐B6dom CTL precursors more abundant than anti‐H‐Y CTL precursors. Dominance resulted from competition for the APC surface between anti‐B6dom and anti‐H‐Y CTL; the crucial difference between the dominant and the dominated MiHA appears to depend on the differential avidity of their respective CTL for APC. The only B6dom epitope thus far identified is the nonapeptide AAPDNRETF presented by H2‐Db. We found that compared with other known Db‐binding peptides, AAPDNRETF is expressed at very high levels on the cell surface, binds to the Db molecule with very high affinity, and dissociates very slowly from its presenting class I molecule. These data indicate that one cannot predict which MiHA will be dominant or dominated based simply on their respective immunogenicity when presented on separate APC. Indeed, the avidity of T cell/APC interactions appears to determine which antigen(s) will trigger T cell responses when numerous epitopes are presented by the same APC.
High and low doses of haptens dictate whether dermal or epidermal antigen‐presenting cells promote contact hypersensitivity
Authors: Bacci Stefano , Alard Pascale , Dai Ruoping , Nakamura Toshiaki , Streilein J. Wayne
Published: March 2007

In the induction of contact hypersensitivity (CH) to an epicutaneously applied hapten, we have previously proposed that low doses of hapten sensitize primarily through epidermal Langerhans' cells (LC), whereas high doses rely largely on dermal antigen‐presenting cells (APC). To examine this issue further, we applied either high or low doses of dinitrofluorobenzene (DNFB) epicutaneously to mice. We observed reduced LC density at the site after 12 h (nadir), which returned to normal levels at 24 h only after a low dose of hapten. When a low dose of an unrelated hapten, oxazolone, was painted on skin that had been painted 12 h previously with high dose of DNFB, oxazolone‐specific CH was impaired. When grafts of whole skin, dermis alone, and epidermis alone prepared from skin painted 2 h previously with low or high doses of DNFB were placed onto naive, syngeneic mice, CH was induced by whole skin after both types of doses, by epidermis only after a low dose, and by dermis only after high dose. When epidermal cell suspensions were derivatized in vitro with low or high doses of DNFB, only cells exposed to a low dose induced proliferation of hapten‐specific T cells. Thus, only a low dose of hapten reveals the APC functions of LC without the participation of dermal APC.
Sequence analysis of human IgVH genes indicates that ileal lamina propria plasma cells are derived from Peyer's patches
Authors: Dunn‐Walters Deborah K. , Isaacson Peter G. , Spencer Jo
Published: March 2007

The origin of human ileal lamina propria (LP) plasma cells has been investigated using a technique based on microdissection of cells from immunohistochemically stained tissue sections. We have sequenced rearranged IgVH4.21, IgVH 5 family and IgVH6 genes from Peyer's patch germinal center (GC) cells and plasma cells from the ileal LP. Clonally related cells were identified by comparison of their complementarity determining region (CDR) 3 sequences and patterns of somatic mutation. We observed Ig genes from B cells in the GC of Peyer's patches which were related to Ig genes from plasma cells in the ileal LP, demonstrating that clonally related cells span these sites. In addition, groups of clonally related, but diversified plasma cells were common in the lamina propria. All Ig genes isolated from LP plasma cells were heavily mutated. The distribution of mutations in the CDR of the Ig heavy chain variable region (VH) genes which were considered to be the expressed alleles in LP plasma cells was consistent with an affinity‐matured response. These observations provide compelling evidence for the origin of human ileal plasma cells from the Peyer's patches.
The CD94 and NKG2‐A C‐type lectins covalently assemble to form a natural killer cell inhibitory receptor for HLA class I molecules
Authors: Carretero Marta , Cantoni Claudia , Bellón Teresa , Bottino Cristina , Biassoni Roberto , Rodríguez Antonio , Pérez‐Villar Juan J. , Moretta Lorenzo , Moretta Alessandro , López‐Botet Miguel
Published: March 2007

CD94, a type II membrane protein containing a C‐type lectin domain, has been shown to be involved in natural killer (NK) cell‐mediated recognition of different HLA allotypes. The inhibitory form of the CD94 receptor has recently been identified by the specific monoclonal antibody (mAb) Z199. Herein, we demonstrate that the inhibitory receptor is in fact a complex formed by the covalent association of CD94 with the NKG2‐A molecule (Mr ∼ 43 kDa), another member of the C‐type lectin superfamily, and that Z199 mAb specifically recognize NKG2‐A molecules. Although the NKG2‐A‐encoding cDNA has been known for several years, the corresponding protein and its possible function remained undefined. Moreover, we show that the NKG2‐B protein, an alternatively spliced product of the NKG2‐A gene, can also assemble with CD94. Remarkably, both NKG2‐A and NKG2‐B proteins contain cytoplasmic immunoreceptor tyrosine‐based inhibitory motifs (ITIM). This may provide the molecular basis of the inhibitory function mediated by the CD94/NKG2‐A receptor complexes.
Binding of a soluble p70 killer cell inhibitory receptor to HLA‐B*5101: Requirement for all three p70 immunoglobulin domains
Authors: Rojo Susana , Wagtmann Nicolai , Long Eric O.
Published: March 2007

Lysis of target cells by natural killer (NK) cells can be prevented by killer cell inhibitory receptors (KIR) specific for major histocompatibility complex class I molecules. Functional studies have identified two distinct p58 KIR, each reactive with a different group of HLA‐C allotypes, and distinct p70 KIR specific for some HLA‐B or HLA‐A allotypes. The NK specificities for each group of HLA‐C allotypes have been reproduced by direct binding of recombinant soluble p58 molecules. Here, we show that a soluble p70 KIR binds to HLA‐B*5101, but not to HLA‐A or HLA‐C molecules. Truncated soluble forms of the HLA‐B*5101‐specific p70 KIR, including one with two immunoglobulin (Ig) domains reactive with a monoclonal antibody that blocks p70 KIR function, did not bind to HLA‐B*5101, indicating that all three Ig domains are required for binding.
Multiple sites of post‐activation CD8+ T cell disposal
Authors: Wack Andreas , Corbella Paola , Harker Nicola , Crispe I. Nicholas , Kioussis Dimitris
Published: March 2007

Antigen‐triggered activation of T cells leads to a sequence of differentiation steps including up‐regulation of activation markers, blast formation, proliferation, delivery of effector functions, and ultimately apoptosis. It is still controversial in which anatomical site activation‐induced apoptosis and elimination of T cells occur. To address this question, we used mice transgenic for a T cell receptor (F5) specific for an influenza virus nucleoprotein peptide (NP68) presented on the major histocompatibility complex H‐2 Db molecule. Accumulation and apoptosis of T cells was studied using terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labeling in situ combined with immunohistology after intraperitoneal injection of the cognate peptide into F5 mice which are wild type or deficient for Rag‐1. After 4 days of peptide treatment, large perivascular infiltrations of CD8+ cells were observed in liver, lung, and kidney of F5 mice. CD8+ cell numbers were also increased in skin and small intestine, but not in brain or heart muscle of peptide‐treated animals. The infiltrating CD8+ cells show an increased percentage of apoptosis in liver, lung and, most strikingly, the kidney. These data suggest that in the F5 system, T cell disposal after activation occurs in a number of organs. Essentially identical findings were obtained in Rag‐1+/+ and Rag‐1‐/‐ F5 mice, suggesting that the deletion mechanism did not involve other T or B cells.
Assembly of an abundant endogenous major histocompatibility complex class II/peptide complex in class II compartments
Authors: Morkowski Stanislaw , Raposo Graça , Kleijmeer Monique , Geuze Hans J. , Rudensky Alexander Y.
Published: March 2007

To identify the intracellular site(s) of formation of an endogenous class II/peptide complex in a human B cell line, we employed kinetic pulse‐chase labeling experiments followed by subcellular fractionation by Percoll density gradient centrifugation and immunogold labeling on ultrathin cryosections. For direct demonstration of assembly of such complexes, we used the monoclonal antibody YAe, which detects an endogenous complex of the mouse class II molecule I‐Ab with a 17‐amino acid peptide derived from the α chain of HLA‐DR (DRα52–68). We show that in human B lymphocytes, these class II/peptide complexes assemble and transiently accumulate in major histocompatibility complex class II‐enriched compartments before reaching the cell surface.
Dimerization of Fab fragments enables ready screening of phage antibodies that affect hepatocyte growth factor/scatter factor activity on target cells
Authors: Zaccolo Manuela , Griffiths Andrew P. , Prospero Terence D. , Winter Greg , Gherardi Ermanno
Published: March 2007

A number of applications of antibodies in diagnosis and therapy require multivalent reagents either because of the polymeric nature of the antigens or because biological activity depends on an effect on the formation of homodimeric species. Here, we report a procedure for mass screening of phage‐derived monomeric antibody fragments that depend on valency for activity. As a model system, a set of 13 phage‐derived human Fab fragments were first selected against mouse and human recombinant hepatocyte growth factor/scatter factor (HGF/SF), a high molecular weight polypeptide growth factor related to the blood protease plasminogen and involved in development and cancer. These Fab fragments were subsequently screened for an effect on HGF/SF activity either as monomeric fragments or after dimerization with a monoclonal antibody (9E10) directed against a peptide tag on the fragments. Fab were identified that either inhibited or enhanced HGF/SF activity on target cell lines, but dimerization was required for this effect. The approach proposed should facilitate mass screening of phage‐derived antibody fragments that depend on multiple valency for activity.
Regulation of I‐309 gene expression in human monocytes by endogenous interleukin‐1
Authors: Selvan Rathinam S. , Zhou Liang‐Ji , Krangel Michael S.
Published: March 2007

Activated human monocytes are a source of numerous γ‐chemokines. The present study was conducted to determine whether these cells produce the human γ‐chemokine I‐309 and to compare the induction requirements of I‐309 to those of other γ‐chemokines. We demonstrate that appropriately stimulated adherence‐purified human peripheral blood monocytes express I‐309 transcripts and secreted I‐309 protein. Two stimuli, immobilized IgG and lipopolysaccharide (LPS), synergize strongly to induce I‐309 gene expression. We further demonstrate that the production of endogenous interleukin (IL)‐1α plays a crucial role in I‐309 induction. Thus, neutralization of endogenous IL‐1α using an anti‐IL‐1α antiserum inhibits the induction of I‐309 transcripts in response to stimulation with immobilized IgG and LPS, and exogenous IL‐1α or IL‐1β induces I‐309 transcripts in monocytes stimulated with immobilized IgG. Immobilized IgG and LPS have the opposite effect on monocyte chemoattractant protein‐1 (MCP‐1) gene expression, in that the induction observed with either stimulus alone is diminished using the two stimuli in combination. Furthermore, endogenous and exogenous IL‐1 can be either stimulatory or inhibitory for MCP‐1 gene expression depending on other signals delivered to the monocytes. Immobilized IgG and LPS synergize to induce macrophage inflammatory protein‐1α transcripts, but endogenous IL‐1 does not play a significant role. Thus, each of these γ‐chemokine genes is under distinct regulatory control in human monocytes.
Qualitatively distinct signaling through T cell antigen receptor subunits
Authors: Jensen Wayne A. , Pleiman Christopher M. , Beaufils Phillippe , Wegener Anne‐Marie K. , Malissen Bernard , Cambier John C.
Published: March 2007

T cell antigen receptors (TCR) contain several subunits including CD3γ, δ, and ϵ, and TCRζ and η which are capable of mediating signal transduction. It is unclear whether the signaling function of these subunits is completely redundant. To assess the relative signaling capabilities of TCR subunits, we compared proximal events in signal transduction by wild‐type TCR complexes and TCR devoid of functional ζ subunits, as well as chimeric receptors containing the cytoplasmic domains of TCRζ or CD3ϵ. Results demonstrate that in BW5147 wild‐type TCR, tail‐less ζ TCR, CD3ϵ, and TCRζ transduce signals leading to tyrosine phosphorylation of similar sets of cellular substrates, including the receptor subunits, Fyn, ZAP‐70, and phospholipase Cγ1 (PLCγ1). Surprisingly, unlike wild‐type TCR, tail‐less ζ TCR, and CD3ϵ, TCRζ was incapable of transducing signals resulting in inositol triphosphate (IP3) generation or intracellular free calcium ([Ca2+]i) mobilization. These data indicate that tyrosine phosphorylation of PLCγ1 is not sufficient to drive IP3 production and [Ca2+]i mobilization. Most importantly, data presented indicate that TCRζ and CD3ϵ engage partially distinct signaling pathways.
Differences in mutational patterns between rheumatoid factors in health and disease are related to variable heavy chain family and germ‐line gene usage
Authors: Børretzen Marie , Chapman Caroline , Natvig Jacob B. , Thompson Keith M.
Published: March 2007

The sequences of the heavy chain variable (VH) segment and dissociation constants (Kd) of 14 IgM rheumatoid factors (RF) derived from 11 different germ‐line gene segments from five healthy immunized donors (HID) are described. We extend a previous analysis of two clones from one donor using only the germ‐line segment DP‐10. In the present study, the mutation patterns of these new RF and the two earlier reported HID RF clones are analyzed in relation to VH family, germ‐line origin, and Kd. The panel of HID RF is further compared with 33 previously described IgM RF from patients with rheumatoid arthritis (RA). There is a high rate of mutation in the panel of HID RF (mean of ten mutations/VH). RF originating in RA patients have a comparable mutation rate (mean of 11 mutations/VH), suggesting that hypermutation of IgM RF is not disease related. The HID RF have, however, a significantly lower affinity for IgG than the RA RF. We found that the structural basis of the differences between HID and RA RF is related to VH family usage. RF of the VH1 family use very similar germ‐line genes in HID and RA patients. HID RF of the VH1 family have, however, a low ratio of replacement‐to‐silent (R : S) mutations of only 0.41 in the heavy chain complementarity region (CDRH) 1 and 2. This is statistically significantly lower than the corresponding ratio of 3.14 in the VH1 RA RF. In contrast, RF of the VH3 family from HID and RA patients have very similar R : S ratios of 1.75 and 1.71 in CDRH1 and 2, respectively. The VH3 RA RF are, however, predominantly encoded by genes not encoding any HID RF. Thus, both repertoire differences and hypermutation resulting in significantly lower R : S ratios can be observed in RF from HID compared with RA RF.
Failure to down‐regulate Bcl‐2 protein in thymic germinal center B cells in myasthenia gravis
Authors: Shiono Hiroyuki , Fujii Yoshitaka , Okumura Meinoshin , Takeuchi Yukiyasu , Inoue Masayoshi , Matsuda Hikaru
Published: March 2007

The most unusual characteristic of myasthenia gravis (MG) is that the thymus has germinal centers (GC). Cultured thymic lymphocytes from MG patients spontaneously produce anti‐acetylcholine receptor antibodies, indicating that autoreactive B cells have escaped negative selection. To investigate the underlying mechanism, we examined the expression of the apoptosis‐related protein Bcl‐2 in GC B cells (defined as CD19+ CD38+ cells) in the thymus in 14 MG patients using three‐color flow cytometry. GC in MG patients did not show the normal down‐regulation of Bcl‐2 (the frequency of Bcl‐2+ GC B cells in the MG thymus and in control tonsils 54.3 ± 16.2% versus 20.6 ± 8.0%; mean ± SD, p < 0.0001). In contrast, Bcl‐2 in GC in the mediastinal lymph nodes from four patients was down‐regulated to a relatively normal level. Using the terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labeling (TUNEL) method to detect DNA fragmentation in situ, the frequency of TUNEL+ cells in GC in the MG thymus was lower than in control tonsils. These results suggest that autoreactive B cells which normally undergo apoptosis in GC may survive because of Bcl‐2 up‐regulation in this unusual location.
Engagement of a T cell receptor by major histocompatibility complex irrespective of peptide
Authors: Rupert Vessey S. J. , Barouch Dan H. , McAdam Stephen N. , Tussey Lynda G. , Davenport Miles A. , O'Callaghan Christopher A. , Bell John I. , McMichael Andrew J. , Jakobsen Bent K.
Published: March 2007

T cell receptors (TCR) identify target cells presenting a ligand consisting of a major histocompatibility complex molecule (MHC) and an antigenic peptide. A considerable amount of evidence indicates that the TCR contacts both the peptide and the MHC components of the ligand. In fully differentiated T cells the interaction between the peptide and the TCR makes the critical contribution to eliciting a cellular response. However, during the positive selection of thymocytes the contribution of peptide relative to MHC is less well established. Indeed it has been suggested that the critical interaction for positive selection is between the TCR and the MHC molecule and that peptides can be viewed as either allowing or obstructing this contact. This predicts that a given TCR is capable of engaging multiple MHC/peptide complexes. In this study a system is described which detects simply engagement of the TCR by MHC/peptide complexes rather than the functional outcome of such interactions. Using this approach the extent to which peptides can influence contacts between the TCR and the MHC molecule has been examined. The results show that the TCR does in fact engage a wide range of ligands in an MHC‐restricted but largely peptide‐independent manner, suggesting that only a few peptides are able to prevent the TCR from contacting the MHC molecule.
Natural killer‐like T cells develop in SJL mice despite genetically distinct defects in NK1.1 expression and in inducible interleukin‐4 production
Authors: Beutner Ulrich , Launois Pascal , Ohteki Toshiaki , Louis Jacques A. , MacDonald H. Robson
Published: March 2007

An unusual subset of mature T cells expresses natural killer (NK) cell‐related surface markers such as interleukin‐2 receptor β (IL‐2Rβ CD122) and the polymorphic antigen NK1.1. These “NK‐like” T cells are distinguished by their highly skewed Vα and Vβ repertoire and by their ability to rapidly produce large amounts of IL‐4 upon T cell receptor (TCR) engagement. The inbred mouse strain SJL (which expresses NK1.1 on its NK cells) has recently been reported to lack NK1.1+ T cells and consequently to be deficient in IL‐4 production upon TCR stimulation. We show here, however, that SJL mice have normal numbers of IL‐2Rβ+ T cells with a skewed Vβ repertoire characteristic of “NK‐like” T cells. Furthermore lack of NK1.1 expression on IL‐2Rβ+ T cells in SJL mice was found by backcross analysis to be controlled by a single recessive gene closely linked to the NKR‐P1 complex on chromosome 6 (which encodes the NK1.1 antigen). Analysis of a panel of inbred mouse strains further demonstrated that lack of NK1.1 expression on IL‐2Rβ+ T cells segregated with NKR‐P1 genotype (as assessed by restriction fragment length polymorphism) and thus was not restricted to the SJL strain. In contrast, defective TCR induced IL‐4 production (which appeared to be a unique property of SJL mice) seems to be controlled by two recessive genes unlinked to NKR‐P1. Collectively, our data indicate that “NK‐like” T cells develop normally in SJL mice despite genetically distinct defects in NK1.1 expression and inducible IL‐4 production.
HLA‐DR‐restricted presentation of purified myelin basic protein is independent of intracellular processing
Authors: Vergelli Marco , Pinet Valerie , Vogt Anne B. , Kalbus Matthias , Malnati Mauro , Riccio Paolo , Long Eric O. , Martin Roland
Published: March 2007

Antigen presentation to CD4+ T cells involves intracellular antigen processing and loading of peptides onto newly synthesized major histocompatibility complex (MHC)‐class II molecules. Some antigens, such as the lipid‐bound, native form of myelin basic protein (LB‐MBP) can also be presented by recycling of cell surface MHC class II molecules. The data reported here demonstrate that a preparation of highly purified, delipidated MBP (HP‐MBP) follows yet another presentation pathway. Similar to LB‐MBP, presentation of HP‐MBP to HLA‐DR1‐restricted T cells was independent of HLA‐DM, of newly synthesized proteins, and of invariant chain expression. However, in contrast to LB‐MBP, presentation of HP‐MBP was also independent of internalization of surface HLA‐DR molecules. The different requirements for the presentation of the two molecular forms of MBP were further confirmed by use of the protease inhibitor E64: presentation of LB‐MBP but not of HP‐MBP was inhibited after treatment of target cells with E64. Furthermore, intact HP‐MPB bound to isolated HLA‐DR molecules in vitro with an association rate that was considerably faster than that of short peptides. These results show that presentation of HP‐MBP is independent of intracellular processing and suggest that it may be presented to T cells by direct binding to surface HLA‐DR molecules.
DNA fragmentation is not the primary event in glucocorticoid‐induced thymocyte death in vivo
Authors: Nakamura Masanori , Yagi Hideki , Ishii Tadashi , Kayaba Shoichi , Soga Hiroyuki , Gotoh Takahiro , Ohtsu Susumu , Ogata Masaki , Itoh Tsunetoshi
Published: March 2007

Thymocyte death has been recognized as one of the best models for studying apoptosis. Our recent study, however, indicated that most thymocytes die without DNA fragmentation and become terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP‐biotin nick end‐labeling‐positive (TUNEL+) only after being phagocytosed by macrophages. In this study, we used histological techniques using the TUNEL method, histochemistry, immunohistochemistry, and transmission electron microscopy as well as flow cytometry to examine in vivo the effect of glucocorticoid (GC), a well‐known agent for inducing thymocyte apoptosis in vitro, on thymocyte death to determine whether or not DNA fragmentation was the first event of GC‐induced thymocyte death. At 2 h and 4 h after GC injection, a large number of cortical thymocytes were TUNEL+. Most TUNEL+ cells were aggregated to form clusters. Double staining of the section showed that the TUNEL+ thymocytes were phagocytosed by acid phosphatase+ and Mac‐2+ macrophages. An ultrastructural study indicated that a far greater number of small pyknotic thymocytes were present in the cortex of the GC‐treated thymus than were observed in the control thymus, that all those pyknotic thymocytes were TUNEL−, and moreover, that at the electron microscopic level, TUNEL+ cells were all phagocytosed by macrophages. Flow cytometric analysis did not detect a single TUNEL+ thymocyte even 4 h after the GC treatment, suggesting that virtually no free dead thymocytes were present after DNA fragmentation. These results indicate that, consistent with our previous findings with normal thymocyte death and B cell death in the germinal centers, DNA fragmentation is not involved in the cell death process of the GC‐induced rapid thymocyte death in vivo.
Fcγ receptor II dependency of enhanced presentation of major histocompatibility complex class II peptides by a B cell lymphoma
Authors: Berg Matthias , Uellner Ruth , Langhorne Jean
Published: March 2007

Here we show that the B cell lymphoma A20.292 is capable of enhanced antigen presentation to CD4+ T cells in the presence of specific antibodies. This enhancement was inhibited by anti‐Fcγ receptor (R) antibodies, suggesting that it might be due to preferential uptake of the antigen/antibody complex through the FcγRII receptor. However, immunoprecipitation studies revealed that the FcR of A20.292 cells was of the B cell type, FcγRIIb1, which is not thought to be able to internalize antigen/antibody complexes via clathrin‐coated pits. It was considered unlikely that A20.292 had an altered form of the B cell FcγR (RIIb1) receptor that enabled internalization, since similar enhancing effects were also observed using an FcγRII− cell line that had been transfected with FcγRIIb1. To reconcile these findings with the expression of FcγRIIb1, it is postulated that immune complexes are concentrated on the cell surface by the FcγRIIb1 and are thus available for preferential uptake by random fluid‐phase endocytosis. This results in more efficient generation of the epitopes recognized by these T cell hybridomas.
A complementarity‐determining region peptide of an anti‐desmosome autoantibody may interact with the desmosomal plaque through molecular mimicry with a cytoplasmic desmoglein 1 sequence
Authors: Gilbert Danièle , Courville Philippe , Brard Frédéric , Joly Pascal , Petit Samuel , Bernardi Elisabeth , Schoofs Alain‐René , Lauret Philippe , Tron François
Published: March 2007

The sera of patients with pemphigus, a group of autoimmune blistering skin diseases, contain autoantibodies directed against components of adhering junctions termed desmosomes. F12, a human monoclonal antibody derived from a pemphigus patient, recognizes an unknown polypeptide of the desmosomal and hemidesmosomal plaques. The third complementarity‐determining region of the F12 heavy chain (VH‐CDR3) was shown to share a four‐amino‐acid sequence (GSSG) with the intracellular domains of desmoglein 1 and bullous pemphigoid antigen 2 which interact with components of, respectively, the desmosomal and hemidesmosomal plaques. Computer modeling of F12 showed that the GSSG sequence protudes inside the antigen‐combining site and thus might be involved in antigen interactions. The GSSG sequence is essential to F12 function, since a peptide containing the VH‐CDR3 inhibited its binding to target antigens while VH‐CDR3 peptides with specific modifications of the GSSG sequence did not. These data allow us to hypothesize that certain autoantibodies produced during the course of an autoimmune disease can behave as adhesion molecules, through the molecular mimicry of the motif involved in protein/protein adhesion, and to propose a new self‐antigen binding mechanism for some autoantibodies.
Co‐stimulatory pathways controlling activation and peripheral tolerance of human CD4+CD28− T cells
Authors: Park Won , Weyand Cornelia M. , Schmidt Dorle , Goronzy Jörg J.
Published: March 2007

Co‐stimulation mediated by the CD28 molecule is considered critical in the activation of CD4+ T cells. In patients with rheumatoid arthritis and infrequently in normal individuals, CD4+ T cells lacking CD28 expression are expanded and contain clonogenic populations. To analyze whether these cells are independent of co‐stimulatory requirements or whether they use co‐stimulatory signals distinct from the CD28 pathway, we have compared CD4+ CD28+ and CD4+ CD28−T cell clones isolated from rheumatoid arthritis patients. Accessory cells supported the induction of CD25 expression as well as of proliferative responses after anti‐CD3 cross‐linking and prevented the induction of anergy in CD4+ CD28− T cell clones. In contrast to CD4+CD28+ T cells, the presence of accessory cells did not enhance the secretion of interleukin (IL)‐2, interferon‐γ, or IL‐4. The co‐stimulatory signals did not involve CD28/CTLA‐4–CD80/CD86 receptor‐ligand interactions. The proliferative response of CD4+CD28− T cells could not be blocked by anti‐CD2, anti‐CD18, and anti‐CD58 antibodies, suggesting that these receptor‐ligand interactions cannot provide CD28− independent co‐stimulation. Our data suggest that CD4+CD28− T cells require co‐stimulatory signals for optimal induction of cell growth and CD25 expression as well as for the prevention of anergy. The co‐stimulatory receptor‐ligand interaction is independent of the CD28 pathway and may be involved in the oligoclonal expansion of the CD4+ CD28− T cell subset in rheumatoid arthritis.
L‐selectin is not essential for naive CD4 cell trafficking or development of primary responses in Peyer's patches
Authors: Bradley Linda M. , Malo Mary E. , Tonkonogy Susan L. , Watson Susan R.
Published: March 2007

We showed previously that L‐selectin‐dependent recirculation of naive CD4 cells is essential for development of primary responses in peripheral lymph nodes. Recent studies suggest that L‐selectin is also required for lymphocyte entry into gut mucosal lymphoid tissues that include Peyer's patches and mesenteric lymph nodes. Here we show that anti‐L‐selectin antibody, MEL‐14, inhibited homing of a rigorously purified, homogenous population of naive CD4 cells into both of these tissues as well as peripheral lymph nodes, directly demonstrating a role for this receptor in regulating entry into gut‐associated sites. However, in intact animals, treatment with MEL‐14 resulted in the loss of naive CD4 cells (CD45RBhi, CD44lo) from peripheral lymph nodes but not Peyer's patches, whereas mesenteric lymph nodes were intermediate in this regard. In mice primed by parenteral immunization with keyhole limpet hemocyanin (KLH), primary CD4 responses were readily detected in both Peyer's patches and mesenteric lymph nodes, and were not affected by exposure to MEL‐14. Indeed, similar frequencies of KLH‐specific CD4 cells were recovered from both of these tissues irrespective of MEL‐14 treatment. The results indicate that interactions with L‐selectin can be circumvented to allow entry of naive CD4 cells into Peyer's patches but not peripheral lymph nodes.
The mechanism of specific prolongation of class I‐mismatched skin grafts induced by retroviral gene therapy
Authors: Mayfield Robin Stanton , Hayashi Hideki , Sawada Tokihiko , Bergen Kate , Leguern Christian , Sykes Megan , Sachs David H. , Iacomini John
Published: March 2007

In the present study, we examine the mechanism of specific hyporesponsiveness to major histocompatibility complex (MHC) class I‐mismatched skin allografts induced by retrovirus‐mediated gene transfer of an allogeneic class I gene into syngeneic bone marrow (BM). Using appropriate congenic recombinant mouse strains, we have mapped MHC determinants that are capable of restoring rapid rejection of Kb‐bearing skin grafts. Our results indicate that either a single class I or a single class II alloantigen expressed on skin in association with Kb is able to restore the rapid rejection of Kb ‐mismatched skin grafts. These data suggest that third‐party alloantigens expressed on skin in association with Kb abrogate hyporesponsiveness by providing T cell help. Consistent with this interpretation, spleen cells from mice reconstituted with Kb‐transduced BM were unable to elicit a significant anti‐Kb cytotoxic T lymphocyte response in vitro unless interleukin‐2 was added to the culture medium. Skin graft survival was also analyzed on B10.AKM mice thymectomized 3–4 weeks post‐reconstitution with Kb‐transduced BM. Thymectomy did not result in significantly prolonged survival of B10.MBR skin grafts compared to euthymic controls, suggesting that even early after reconstitution, intrathymic deletion of Kb‐reactive T cells must have been incomplete. Taken together, these data suggest that prolongation of skin allograft survival in this model is controlled at the level of T cell help.
Allergen‐induced recruitment of FcσRI+ eosinophils in human atopic skin
Authors: Barata Luis T. , Ying Sun , Andrew Grant J. , Humbert Marc , Barkans Julia , Meng Qui , Durham Stephen R. , Barry Kay A.
Published: March 2007

We have attempted to identify FcσRI+ eosinophils in cutaneous late‐phase reaction in atopic subjects biopsied at 6, 24 and 48 h after the injection of either allergen or a diluent control. Compared to the diluent sites, allergen‐injected sites had significantly increased numbers of eosinophils, peaking between 6 and 24 h, of which approximately 20–30% expressed mRNA for the α, β, and γ chains of FcσRI, as shown by in situ hybridization. Using either a monoclonal or a polyclonal anti‐α chain antibody, the FcσRIα protein also co‐localized to approximately 50–80% of eosinophils at all time points studied. We also observed a significant correlation (r = 0.89; p = 0.02) between the numbers of FcσRI+ (997+)/EG2+ eosinophils and the magnitude of the late‐phase reaction. Thus, a significant proportion of eosinophils infiltrating the site of allergen‐induced allergic tissue reactions in atopic subjects express FcσRI. The findings show that high‐affinity IgE receptors may play a role in eosinophil secretory processes in vivo.