摘要:

AbstractWe report striking immunodominance in the neutralizing antibody responses of major histocompatibility complex congenic mice to natural infection with influenza virus (H3N2 subtype), as deduced by sequencing the hemagglutinin (HA) genes of monoclonal antibody (mAb)‐selected mutant viruses. A majority of mAb, established from individual BALB/c (H‐2d) mice, select mutant viruses containing the same single amino acid substitution in the membrane distal ectodomain, HA1 198 A→E, whereas changes at either HA1 158 G→E or HA1 198 A→E are selected for by mAb from BALB.K (H‐2k) donors. The structural basis for immunodominance, and potential diversity of progenitor B cells, was investigated by sequence analysis of H and L chain gene rearrangements in mAb specific for HA1 158 or HA1 198. No correlation was found between antibody specificity and VHor VLgene usage, and a minimum of three to six progenitor cells contributed to the individual's repertoire for a single antigenic site. However, in a further analysis of the HA1 158‐specific antibody response of CBA/Ca (H‐2k) donors, there was highly restricted light chain gene usage. Focusing of the immune repertoire to limited regions of the HA molecule during a primary viral infection may be a significant factor in immune pressure for antigenic variation, particularly since there is no evident restriction in the antibody response

ISSN:0014-2980    

DOI:10.1002/eji.1830250702

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractHeme is a non‐protein autoantigen which is ubiquitousin vivo, primarily complexed in various hemoproteins or bound to specialized carrier molecules. Nevertheless, heme is able to stimulate a high frequency of CD4+, class II‐restricted T cells, freshly explanted from unprimed mice, to proliferatein vitro. In this study, we show that heme incorporated into various species of mammalian cytochromec(cytc), including murine cytc, represents a facultative cryptic determinant, able to be recalled only at high doses of native cytc. By contrast, avian cytcis of comparable antigenicity to free heme. Artificially denatured carboxymethylated (CM) mammalian cytcexhibited greatly increased antigenicity, comparable to that of heme and avian cytc, indicating that the crypticity of heme in native mammalian cytcis due to the resistance of the native conformation of this molecule to antigen processing within murine antigen‐presenting cells. Thus, tolerance to the heme group of at least some hemoproteins, may be maintained by the crypticity of the heme, rather than by deletion of hemereactive T cells. Given the high frequency of heme‐reactive T cells in unprimed mice, these findings suggest that heme may become an important modulator during an inflammatory r

ISSN:0014-2980    

DOI:10.1002/eji.1830250703

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractThe glycosylphosphatidylinositol (GPI)‐anchored CD59 protein (human protectin) protects cells against complement‐induced lysis, binds to CD2 and also transduces activation signals within T cells. We have further examined the biochemical signals transduced by CD59 and addressed its role in regard to the CD3‐mediated signaling cascade. We show here that CD59 cross‐linking induces a time‐dependent activation of p56lckand of p70zap(ZAP‐70) in CD3‐positive Jurkat cells, leading to the stimulation of the T cell receptor ζ/ZAP‐70 signaling cascade and interleukin‐2 (IL‐2) synthesis. Cross‐linking of CD59 on peripheral T cells and thymocytes induces tyrosine phosphorylations identical to those seen in Jurkat cells and this is followed by lymphokine production and proliferation. In contrast, only activation of CD59‐associated p56lckoccurs in CD3‐negative Jurkat cells, while IL‐2 production is impaired, consistent with the lack of ZAP‐70 tyrosine phosphorylation observed in these cells. CD59 triggers activation events even in the absence of CD3/T cell receptor expression in Jurkat cells. CD59 cross‐linking synergizes with sub‐optimal doses of phorbol ester for activation of the protein kinase C and of the p42mapk, as shown byin vitrophosphorylation of histone HIIIS and myelin basic protein, respectively, and leads to CD25 but not CD69 expression. In conclusion, at least two signaling pathways are triggered through CD59, the first one involving ZAP‐70 activation and leading to IL‐2 secretion and a second pathway observed in the absence of ZAP‐70 activation leading to CD25 expression. These two pathways are likely to be involved in the modulat

ISSN:0014-2980    

DOI:10.1002/eji.1830250704

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractMost native antigens require digestion by acidic proteases in order to be recognized in the context of major histocompatibility complex class II by T helper cells (Th). We have studied the roles of three different acidic proteases, cathepsin D, cathepsin B and cathepsin L, in the processing of ovalbumin (OVA) for presentation in the context of I‐Ad. We report that digestion of OVAin vitrowith the aspartyl protease cathepsin D generates the epitope OVA322–336, which is recognized by I‐Ad‐restricted OVA‐specific Th in the presence of paraformaldehyde‐fixed antigen‐presenting cells (APC). In contrast, digestion of OVA with the cysteine proteases cathepsin B and L not only failed to generate an epitope, but also destroyed OVA322–336. In the presence of fixed APC expressing I‐Ad, OVA322–336was protected from destructive proteolysis by cathepsin L. These results illustrate the dependence of epitope selection on the intracellular proteolytic environment in APC, and suggest that mechanisms must exist for protection of epitopes from destructive proteolysis in the proc

ISSN:0014-2980    

DOI:10.1002/eji.1830250705

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractRegulation of the development of thymocytes into mature T cells within the thymus is now known to involve antigen‐induced deletion, by apoptosis, of potentially autoreactive thymocytes, and it can be mimicked either by stimulating the T cell receptor (TcR) complex by monoclonal antibody (mAb) or by ionophore‐induced elevation of cytosolic [Ca2+]. To identify signaling pathways employed by the TcR complex of immature thymocytes, we examined the effects of anti‐CD3 and anti‐TcRß constant (c) region mAb, staphylococcal enterotoxin B (SEB) and pharmacological agents on the generation of inositol phosphates through hydrolysis of phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] both in cultured fetal mouse thymic lobes and in the CD4+CD8+immature thymocyte cell line, TM10G. Stimulation of the TcR complex with anti‐CD3 mAb provoked an accumulation of inositol phosphates diagnostic of the occurrence of receptor‐stimulated phosphoinositidase C (PLC) activation. Anti‐TcRCß mAb and SEB provoked smaller but similar responses. The PLC activation evoked by anti‐CD3 mAb was suppressed by inhibitors of receptor tyrosine kinases and was unmodified by protein kinase C activation or elevation of cytosolic [Ca2+]. It thus appears that apoptosis triggered by TcR stimulation is associated with PLC activation by a receptor‐regulated tyrosine kinase. Treatment of thymic lobes or TM10G cells with fluoroaluminate provoked apoptosis of a wider range of thymocyte subtypes and such stimulation also provoked an accumulation of inositol phosphates. The responses to fluoroaluminate were not prevented by inhibitors of tyrosine kinases, suggesting that unidentified GTP‐binding proteins which couple to PLC activation may also be capable of initiating apoptosis by a route independent of the TcR. These results, when considered alongside previous studies of mature T cells, indicate that stimulation of immature thymocytes or of mature T cells through their TcR complex activates the PLC‐catalyzed PtdIns(4,5)P2hydrolysis signaling pathway, and thus that this signaling pathway may be implicated both in provoking apoptosis in immature T cells and in initiating prolif

ISSN:0014-2980    

DOI:10.1002/eji.1830250706

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractSeveral alterations in T cell receptor‐associated signal transduction have been observed following induction of anergy of T helper type 1 (Th1) clones, including a modified intracellular free calcium ([Ca2+]i) response and increased kinase activity associated with the protein tyrosine kinase p59fyn. In the current study, we demonstrate that, although the kinetics of acquisition of both of these signaling alterations correlated with the generation of anergy, a normal calcium response returned within 48 h after removal from the anergizing stimulus, whereas the increased p59fynactivity persisted and the cells remained hyporesponsive. Generation of both the anergic state and the increased p59fynactivity was prevented in the presence of calcium‐free medium, cycloheximide (CHX), or cyclosporin A (CsA), and could be mimicked by the calcium ionophore ionomycin. In contrast, the altered calcium response was inhibited by stimulation in the presence of calcium‐free medium or CsA, but not CHX. Thus, surprisingly, these data suggest that a chronic elevation of [Ca2+]iis proximal to and necessary for the increase in p59fyn‐associated kinase activity observed in anergic Th1 clones. Increased p59fynactivity, but not the altered calcium response, correlates with maintenance of the anergi

ISSN:0014-2980    

DOI:10.1002/eji.1830250707

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractThe CD4 coreceptor interacts with non‐polymorphic regions of major histocompatibility complex class II molecules on antigen‐presenting cells and contributes to T cell activation. We have investigated the effect of CD4 triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that CD4 triggering delivers signals capable of activating the NF‐AT transcription factor which is required for interleukin‐2 gene expression. Whereas different anti‐CD4 mAb or HIV‐1 gp120 could all trigger activation of the protein tyrosine kinases p561ck and p59fyn and phosphorylation of the Shc adaptor protein, which mediates signals to Ras, they differed significantly in their ability to activate NF‐AT. Lack of full activation of NF‐AT could be correlated to a dramatically reduced capacity to induce calcium flux and could be complemented with a calcium ionophore. The results identify functionally distinct epitopes on the CD4 coreceptor involved in activation of the Ras/protein kinase C and c

ISSN:0014-2980    

DOI:10.1002/eji.1830250708

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractThe appearance of peptide‐loaded major histocompatibility complex (MHC) class II molecules at the cell surface depends critically on the invariant chain (Ii). We have studied the influence of Ii on the positive selection of CD4+T cells, mediated by class II molecules expressed on thymic stromal cells. Invariant chain‐deficient mice (Ii°) were crossed with different T cell receptor (TcR) transgenic strains and the emergence of mature CD4 single‐positive thymocytes measured in Ii°/TcR transgenic offspring. Positive selection was nearly absent in Ii°/2B4 mice, which display receptors specific for a moth cytochrome c (MCC) peptide in the context of Ek. In addition, no T cell response was elicited when nontransgenic Ii° animals were injected with this peptide, even though antigenpresenting cells (APC) from such mice were perfectly capable of presenting it, suggesting that selection of the entire anti‐MCC 88‐103 repertoire depends on Ii. Positive selection also appeared strongly reduced in another line of Ii°/TcR transgenic mice (Ii°/BDC2.5). However, in sharp contrast, a third line (Ii°/3A9) exhibited almost normal positive selection of thymocytes displaying the transgene‐encoded receptor. These thymocytes were exported to the periphery; peripheral T cells could respond normally to the appropriate peptidein vitro. The most likely interpretation of these findings is that selection of most CD4+T cells depends on MHC class II complexes loaded with peptide in an Ii‐dependent pathway, but some can be selected on class II complexes that are either loaded along an alternative, Ii‐independent, route or are empty. This is consistent with the involvement of peptide in positive selection of CD4+T cells, for which there exists

ISSN:0014-2980    

DOI:10.1002/eji.1830250709

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractInterleukin (IL)‐5 binds to a cell surface receptor composed of two polypeptide chains, α and β, both belonging to the hemopoietic cytokine receptor family. Mouse cells expressing common mouse β chain (AIC2B) that were transfected with human IL‐5 receptor (R)α cDNA proliferated in response to picomolar concentrations of human IL‐5, indicating that a functional receptor was reconstituted. We show that in these cells, human (h)IL‐5 as well as mouse (m)IL‐3 induce tyrosine phosphorylation of β chain and JAK 2 kinase. Phosphorylated β receptor was co‐precipitated with anti‐JAK 2 antibodies, suggesting that both molecules were physically associated. IL‐5 and IL‐3 also induce cytosolic DNA binding activity as measured by an electrophoretic mobility shift assay using the interferon‐γ responsive region of human Fc γ1 gene DNA element. A deletion mutant of hIL‐5Rα lacking the cytoplasmic part could bind hIL‐5 normally in association with the β chain, but was unable to transmit a biological signal. The cytoplasmic domain was also indispensable for tyrosine phosphorylation and activation of DNA binding proteins. A membrane‐proximal proline‐rich element of the hIL‐5Rα cytoplasmic domain that is conserved among different members of the hemopoietic cytokine receptor family was essential for biological activity. Point mutations in this motif also knocked

ISSN:0014-2980    

DOI:10.1002/eji.1830250710

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY

摘要:

AbstractThe ileal Peyer's patch (PP) in sheep plays a central role in the development and production of B cells. Associated with a tremendous amount of B cell proliferation in this site is the extensive diversification of the Ig repertoire by somatic hypermutation. Very few (<5%) of the B cells produced in the ileal PP differentiate and emigrate; instead, the vast majority of these cells soon die, and we have previously shown that death is associated with apoptosis. When placed in culture, ileal PP B cells die rapidly by apoptosis, such that after 24h, 60 ± 1 % of DNA is fragmented. Here, we show that the extent of this spontaneous B cell apoptosis in culture, as quantitated by DNA fragmentation, was significantly increased in a dose‐dependent manner by the glucocorticoids hydrocortisone or dexamethasone. Furthermore, treatment of lambs with 2–2.5 mg/kg of dexamethasone resulted in a marked increase in the number of apoptotic cells in the ileal PP and an increase in ileal PP B cell DNA fragmentation to 20 ± 6%, compared with 2.4 ± 0.1 % in untreated lambs. Anti‐immunoglobulin (Ig) antibodies also increased the extent of DNA fragmentation in cultured ileal PP B cells. After 24 or 48 h of culture with anti‐Ig (PIg47A), DNA fragmentation was 74 ± 2 % and 75 ± 3 %, respectively. Ileal PP B cells are rescued from apoptosis by agents that activate protein kinase C and increase cytosolic Ca2+, and here we show that this treatment also results in apoptotic rescue in the presence of dexamethasone or anti‐Ig. We speculate that the apoptosis of ileal PP B cellsin situmay be modulated by glucocorticoids and by the cross‐linking of surface Ig. Apoptosis, induced by a signal through surface Ig, may be an important mechanism in the deletion of self‐reactive B cells during the expansion of the Ig repertoi

ISSN:0014-2980    

DOI:10.1002/eji.1830250711

出版商:WILEY‐VCH Verlag GmbH    

年代:1995

数据来源: WILEY