摘要:

AbstractThe heterogeneity of the thymic stroma has made careful characterization of particular thymic stromal cell types difficult. To this end, we have derived a panel of cloned thymic stromal cell lines from simian virus 40 T antigen (SV40‐T antigen) transgenic mice. Based on their analysis with monoclonal antibodies that distinguish among subsets of thymic stroma cells, and on the morphology and ultrastructural features of the different clones, we suggest that our panel includes representatives of the thymic subcapsular cortex or thymic nurse cells (427.1), the deep cortex or cortical reticular cells (1308.1) and the medulla including medullary interdigitating (IDC)‐like cells (6.1.1) and medullary epithelial cells (6.1.7). A fifth cell type of undesignated but apparent medullary origin (6.1.11) was also isolated. All of the cell lines constitutively express the SV40 T antigen transgene and the class I antigens of the major histocompatibility complex (MHC), and they can be induced to express MHC class II antigens upon stimulation with recombinant interferon‐γ (IFN‐γ). These cell lines elaborate a factor(s) that induces the proliferation of cells from the fetal liver and bone marrow, but not from the neonatal thymus. A factor(s) elaborated by the 1308.1 cell line also induces the proliferation of fetal thymocytes in the absence of mitogens, phorbol esters or calcium ionophore which is augmented with the addition of recombinant interleukin‐2 (IL‐2). Analysis by reverse transcription polymerase chain reaction with primers for some mouse cytokines reveals that each of these cell lines contain granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) transcripts and that 1308.1,6.1.1 and 6.1.7 produce IL‐6 mRNA. Cell lines 1308.1 and 6.1.1 also produce IL‐7; 6.1.1 produces IL‐lβ and tumor necrosis factor (TNF)‐α while the 427.1 cell line produces IL‐5 and IFN‐γ mRNA. None of the cell lines tested express the IL‐2 receptor, IL‐2, IL‐3, IL‐4, TNF‐β or macrophage inflammatory proteins mRNA. Conditioned medium (CM) from 1308.1 and 6.1.11 induced differentiation of cells purified from the mouse fetal liver into granulocytes; 1308.1 CM also induced differentiation of the mouse hematopoietic stem cell line 32DC13(G) suggesting that the CM contains granulocyte (G)‐CSF activity. Each cell line produces GM‐CSF but the greatest activity is associated with 1308.1 and 6.1.11 CM. The availability of these well‐characterized, functional, cloned thymic stromal cells will allow a more detailed analysis of the role of e

ISSN:0014-2980    

DOI:10.1002/eji.1830230602

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractWe used the human processing defective cell line 174CEM.T2 (T2) to identify potential cytotoxic T lymphocyte (CTL) epitopes of human proteins. Exogenously added peptides can increase the number of properly folded HLA‐A2.1 molecules on the cell surface of T2 cells, as shown by immunofluorescence measurements using the mouse monoclonal antibody BB7.2 (anti‐HLA‐A2.1) and fluorescein isothiocyanate‐labeled goat anti‐mouse F(ab')2 antibody. The peptides were selected on the basis of a computer score derived from the recently described HLA‐A2.1 specific motif. Analysis of the influenza matrix protein showed that 15 out of 35 high‐scoring peptides up‐regulate the expression of HLA‐A2.1 molecules on theT2 cell surface. The combination of the computer scoring program and an immunofluorescence‐based peptide binding assay allows rapid detection of potential

ISSN:0014-2980    

DOI:10.1002/eji.1830230603

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have established IgG rheumatoid factor (RF)‐secreting hybridoma cell lines from the synovial tissues of three patients from whom we have previously characterized several IgM RF. The IgG RF bind human and rabbit IgG and form intracellular complement‐fixing complexes indicative of a self association processin vivo. Nucleotide sequence analysis revealed that two IgG RF used VHIII gene segments, while one used a VHI gene segment. The VLgene usage consisted of a Vχ1, a Vλ2 and a Vχ4/Vχ6 hybrid, confirming our previous findings that many different VLgenes can contribute to RF specificity. Although the IgG RF used VHgenes from the same families that dominated the IgM RF response, two of the actual gene segments employed were not found among the IgM RF. In contrast to IgM RF, which in general were not very mutated, the IgG RF showed somatic mutations characteristic of an antigen‐driven immune

ISSN:0014-2980    

DOI:10.1002/eji.1830230604

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractPrevious work has shown that neutralization of physiologically secreted interferon(IFN)‐γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN‐γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90‐kDa IFN‐γ receptor (IFN‐γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN‐γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW‐PBL) and its expression increases two‐ to threefold on alloactivated NW‐PBL. IFN‐γR protein is poorly expressed on the membrane of resting CD3+cells, but up‐modulates after 3‐day MLR and sharply down‐modulates at day 6. Both the p55 and the p75 chains of interleukin‐2 receptor (IL‐2R) were shown to up‐modulate in parallel with IFN‐γR, whereas they were still highly expressed at day 6. After alloactivation, IFN‐γ and IL‐2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN‐γR and IL‐2R begin to up‐modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN‐γR membrane up‐modulation, and IFN‐γ and IL‐2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN‐γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis th

ISSN:0014-2980    

DOI:10.1002/eji.1830230605

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractT lymphocytes from patients with multiple sclerosis (MS) recognize multiple myelin basic protein (MBP) epitopes. This situation complicates the design of specific immunotherapies. We investigated to which extent the T cell response to MBP is heterogeneous in single subjects in terms of preferentially recognized regions of the molecule, major histocompatibility complex (MHC) restriction, and stability over time. From each of nine patients with MS, a minimum of six MBP‐specific T lymphocyte lines (TLL) were assayed for the proliferative response to a panel of overlapping peptides, encompassing the whole MBP. Predominant Tcell recognitions of distinct MBP regions were present in three patients, all HLA‐DR2+, independently of the clinical features of their disease. Tcell reactivity was preferentially directed to residues 16‐38 in one patient. In this case the response was also stable over time, during different phases of the disease. Predominant reactivity to residues 86‐99 was detected in the two other DR2+patients. In each of the patients with other HLA‐DR haplotypes (DR2−), as well as in three DR2+non‐MS donors, the Tcell response to MBP appeared to be considerably more heterogeneous. The HLA restriction element varied among TLL recognizing the same MBP region, even when raised from the same individual. The genomic HLA typing, performed on the DRB1 and DRB5 genes in the DR2+subjects, showed no obvious correspondence between preferential responses to regions of MBP and HLA‐DR2 subtypes. In this context, a simple, new method for the genomic typing of the HLA‐DRB1 gene in individuals with the HLA‐DR2 serological specificity is also described.We conclude that predominant and stable T cell responses to a single MBP region can be detected in some patients with MS. In these individuals, the MHC restriction of the T cell recognition of predominant regions appears to be variable. Polymorphisms of the HLA‐DR2 gene products alone do not account for the selection of the domin

ISSN:0014-2980    

DOI:10.1002/eji.1830230606

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractInterleukin‐1α (IL‐1α) has been implicated in the pathogenesis of infectious, autoimmune and inflammatory diseases. There is, however, very little information on the cis‐acting sequences involved in IL‐1α regulation or whether there is any variation in the structure of the gene. It is known that intron 6 of IL‐1α shows a 5 × 46 bp tandem repeat in the genomic sequence. We have studied this region of the gene. Amplification by polymerase chain reaction showed different sized products from different individuals, most being of higher molecular weight than the expected size of 620 bp. Sequencing demonstrated that the polymorphism was due to a variable number of repeats of the 46 bp sequence. This was confirmed by restriction fragment length analysis of genomic DNA. Altogether, 72 unrelated individuals were tested and 6 alleles ranging from 5 to 18 repeats were found, the most frequent allele (62%) containing 9 repeats. This polymorphism may be of interest in gene function, since each repeat contains three potential binding sites for transcriptional factors: an SP1 site, a viral enhancer element and a glucocorticoid‐responsive element. The latter, at least, demonstrates site‐specific protein binding by electromobility shift assay. The functional significance of the polymorphism and its allelic frequency in inflammatory and autoimmune diseases are currently u

ISSN:0014-2980    

DOI:10.1002/eji.1830230607

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractNegative selection in thymus occurs by apoptosis in CD4+CD81cells. These immature thymocytes are readily killed, bothin vitroandin vivo, by glucocorticoid treatment. Increased levels of intracellular cAMPin vitroalso induce apoptosis of thymocytes and T cell receptor (TcR) stimulation potentiate cAMP responses through receptors linked to adenylic cyclase. Presently, we have tested the possibility that TcR‐mediated apoptosisin vivomay require the glucocorticoid receptor (GR) as a downstream, intracellular mediator. Use of the GR antagonist RU‐486, 24 h before and simultaneous with, anti‐CD3 or 5′‐(N‐ethyl)‐carboxamide‐adenosine (NECA) treatment, resulted in a selective inhibition of CD4+CD8+thymocyte death. In addition, a low dose of glucocorticoid potentiated thymocyte death induced by anti‐CD3 monoclonal antibodies. These data support a model in which thymic negative selection depends on a defined set of transduction signals which potentiate the GR to become responsive to endogenous levels

ISSN:0014-2980    

DOI:10.1002/eji.1830230608

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractThe CD28 antigen has been recently demonstrated to be a costimulatory molecule and is expressed by almost all thymic and peripheral T cell receptor (TcR) αδ+and γλ+cells in the mouse system. We show here that expression of CD28 is heterogeneous among murine intestinal intraepithelial lymphocytes (IEL). Whereas some TcR αβ‐expressing IEL subsets such as CD4+8−and CD4−8α+β+cells express CD28 at the same levels as their phenotypic counterparts in lymph node, other subsets of TcR αβ cells (including CD4−8α+β−and CD4+8αβ+β cells) as well as TcR γλ+IEL fail to express CD28. Parallel experiments using aged BALB/c‐nu/nu mice indicated that CD28 expression patterns among IEL are quite similar to those of normal BALB/c mice. Furthermore, forward light scatter analysis showed that CD28−cells are considerably larger than CD28+cells in the gut, although cycling cells were rare in both subsets. Finally CD28−cells in the gut did not proliferate or produce IL‐2 upon stimulation by anti‐CD3 monoclonal antibodies (mAb) and phorbol 12‐myristate 13‐acetate, whereas CD28+cells in the gut and lymph nodes responded to these stimuli. The response of the CD28+cells was enhanced by anti‐CD28 mAb. These results suggest that CD28−IEL (CD4‐8α+β−cells, and some CD4+8α+β−cells) may follow a different developmental pathway from that of CD28+IEL in a thymus‐independent environment, and that expression of CD28 correlates with responsiv

ISSN:0014-2980    

DOI:10.1002/eji.1830230609

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractWe have examined the T cell receptor (TcR) expression of clones specific for epitopes of mycobacterial 65‐kDa heat‐shock protein (hsp65) in the context of two different HLA molecules, and used this system as a model to assess the selection of T cells responsive to this antigenin vivo.DR3‐restricted clones were raised from both the synovial fluid (SF) and peripheral blood (PB) of a patient with reactive arthritis in three separate cloning events. Five of five SF‐derived clones tested expressed either Vβ5.2 or a closely related β chain, Vβ5.6.The α chains expressed by Vβ5.2+and Vβ5.6+clones were from different families, Vα2.4 and Vα23.2, respectively. Nine of ten clones derived from two cloning procedures on PB taken 3 years later also expressed either Vβ5.2 or Vβ5.6. This suggests that the TcR repertoire for recognizing this major histocompatibility complex/peptide complex is relatively restricted and favors the use of Vβ5. Conservation of the β chain third complementarity‐determining region (CDR3) sequence was not evident, however. Sequencing α and β chains of representative Vβ5.2+and Vβ5.6+PB‐derived clones revealed TcR which were identical to those utilized by the SF‐derived clones, showing that the repertoire for recognition of this antigen is stable over time.Similar studies of TcR expression were carried out on hsp65‐specific, DP4‐restricted clones derived from the SF of a patient with rheumatoid arthritis by two independent cloning procedures. There was conservation of a chain usage, since all clones expressed a member of the Vαl family, but again CDR3 sequence conservation was not apparent, β chain usage was not restricted since different clones expressed Vβ6.7, Vβ22.3 and Vβ12. Subtle differences in epitope specificity were detected for two clones with differing TcR. Once more, T cell clones with identical α and β TcR chains were obtained from the separate cloning procedures, suggesting oligoclonalty of T cells with this

ISSN:0014-2980    

DOI:10.1002/eji.1830230610

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY

摘要:

AbstractThe expression of complement receptor 2 (CR2) has been demonstrated in established HTLV‐1‐transformed cell lines and in 12 studiedde novoinfected peripheral blood lymphocytes cultures, using 2 HTLV‐1 sources. The simultaneous detection of CR2 and HTLV‐1 antigens in both co‐cultivated and supernatant‐infected peripheral blood lymphocytes suggest that the increased CR2 expression is in tandem with the increasing HTLV‐1 antigen expression. CR2 up‐regulation seen during polyclonal activation is presumably in response to a viral protein, although a cellular factor has not been ruled out. Increasing CR2 expression during early infection suggests its possible involvement in selection or development of subsequent transformation events. Variable levels of CR2 in immortalized cell lines argue against its obligate expression of function in the maintenance of the transformed state. The expression of CR2 in cellular activation of T cells may be stage restricted. This study also expands the cellular distr

ISSN:0014-2980    

DOI:10.1002/eji.1830230611

出版商:WILEY‐VCH Verlag GmbH    

年代:1993

数据来源: WILEY