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OBJECTIVE: To investigate the expression of human cartilage (HC) gp-39, a possible autoantigen in rheumatoid arthritis (RA), in peripheral blood and synovium, to characterize its cellular source, and to analyze correlations with clinical features. METHODS: The expression of HC gp-39 in synovium and peripheral blood mononuclear cells (PBMC) was assessed by immunohistochemistry and flow cytometry. Synthesis and secretion were investigated by both reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: PBMC expressing HC gp-39 were increased in RA patients compared with spondylarthropathy patients (P = 0.0029) and with healthy control subjects (P = 0.0013). HC gp-39+ cells were also slightly overrepresented in RA synovium (P = 0.01). In both blood and synovium, HC gp-39+ cells were identified as CD14dim,CD16+ monocytes, a phenotype which can differentiate from classic CD14++ monocytes by maturation in vitro. HC gp-39 messenger RNA was detected in RA synovium and PBMC, and PBMC secreted HC gp-39 in vitro. The number of HC gp-39+ PBMC correlated with serum levels of C-reactive protein (r = 0.39, P = 0.003) and HC gp-39 (r = 0.52, P = 0.014). HC gp-39 expression in RA synovial lining correlated with joint destruction (r = 0.77, P < 0.001). CONCLUSION: CD16+ monocytes, a cellular source of HC gp-39 in vivo, are overrepresented in both RA peripheral blood and synovial tissue. The presence of HC gp-39+ cells in RA synovium is correlated with the degree of joint destruction. These data support a role of these cells in the local autoimmune response that leads to chronic inflammation and joint destruction.

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OBJECTIVE: To analyze the CD4+ T cell responses to the human cartilage antigen glycoprotein-39 (HCgp-39) in the context of rheumatoid arthritis (RA)-associated (DRalphabeta1*0401) and nonassociated (DRalphabeta1*0402) HLA class II molecules. METHODS: Large numbers of HCgp-39-specific T cell hybridomas were generated following immunization of HLA-DR4/human CD4 transgenic, murine major histocompatibility complex class II deficient mice with native HCgp-39. Fine epitope mapping of DRalphabeta1*0401-and DRalphabeta1*0402-restricted T cell hybridomas was performed using overlapping synthetic peptides. Antigen-specific cytokine production by lymph node T cells was evaluated after immunization with native antigen. Proliferative T cell responses of healthy human subjects were compared with the T cell responses of patients with active RA using HCgp-39 epitopes defined in HLA-DR4 transgenic mice. RESULTS: CD4+ T cells from DRalphabeta1*0401 and DRalphabeta1*0402 transgenic mice identified completely different immunodominant peptide epitopes of HCgp-39, and this was not explained by known DR4-binding motifs or direct peptide-binding studies. DRalphabeta1*0401-restricted, antigen-specific T cells produced significantly more interferon-gamma and tumor necrosis factor a in response to HCgp-39 than did T cells from DRalphabeta1*0402 transgenic mice. Finally, HCgp-39 peptides defined in DRalphabeta1*0401 transgenic mice stimulated T cells from HLA-DR4 positive human subjects and RA patients, but not T cells from HLA-DR4 negative individuals. CONCLUSION: T cell epitopes of HCgp-39 that were defined in HLA-DR4 transgenic mice stimulated T cells from human subjects carrying RA-associated HLA-DR4 alleles. HLA-DR4 molecules may influence the disease process in RA both by presentation of selected peptide epitopes and by promoting the production of proinflammatory cytokines in synovial joints.

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Monoclonal antibodies (mAb) specific for the clonotype of an autoreactive T cell may be useful reagents in the modulation of autoimmune disease. We have previously reported the generation of a set of mAb specific for the clonotypic structure of a human T-cell clone recognizing an epitope of human cartilage gp-39. This glycoprotein was recently identified as a candidate autoantigen in rheumatoid arthritis. Here, we demonstrate for the first time that small amounts of immobilized anticlonotype mAb can induce anergy in the autoreactive clone. Following the anergic stimulus, T cells failed to proliferate upon restimulation as a result of a lack of interleukin-2 (IL-2) gene transcription. In addition, a diminished interferon-gamma (IFN-gamma) production was found. Our data indicate that anergy was not a result of T-cell receptor (TCR) downmodulation or the absence of free TCR. The anergic state was induced independent of costimulation or the presence of IL-2 and no protein synthesis was required for the induction of anergy. Anticlonotype mAb-induced anergy was prevented by cyclosporin A, suggesting that active signalling via the calcium/calcineurin pathway was required for the induction of anergy. In coculture experiments, anergic T cells were found to suppress the response of reactive cells from the same clone. This bystander suppression led to 90% inhibition of peptide-induced proliferation. Together, these findings suggest that mAb to the clonotypic structure of autoreactive T cells may be suitable reagents for the functional inactivation of these T cells in autoimmune diseases.

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OBJECTIVE: To study at a molecular level the clonality of interleukin 2 (IL-2) expanded T cell lines derived from rheumatoid nodules. Such cell lines were reported in earlier studies with flow cytometry and antiidiotypic monoclonal antibodies (MAb) to be obligoclonal. METHODS: T cell lines were derived from rheumatoid nodules in 2 patients with rheumatoid arthritis (RA) and expanded in medium containing IL-2. Clonality was assessed by flow cytometry and T cell receptor (TCR) idiotype specific Mab and by polymerase chain reaction with primers for V alpha and V beta gene families. Sequence analysis was performed in selected cell lines. RESULTS: In one patient, one cell line was identified with marked overexpression of V alpha 2 cells. Eleven V alpha 2 CDR3 sequences were derived from this cell line: 8 of these clones had an identical CDR3 sequence and one other clone showed a related sequence. Five cell lines derived from a second patient displayed a marked clonal bias to V beta 8 cells. One cell line with strong V beta 8 expression was chosen for further sequence analysis. Twelve V beta 8 sequences were obtained; 11 showed identical CDR3 sequences. CONCLUSION: Molecular analysis of TCR rearrangements in IL-2 expanded T cell lines from rheumatoid nodules strongly suggests that in situ T cell activation is related to classical antigen induced immune activation.

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OBJECTIVE: To identify a cartilage-derived autoantigen that is relevant to the rheumatoid arthritis (RA) disease process. METHODS: A DR4 (DRB1*0401) peptide binding motif was used for the selection of potential self reactive peptides within human cartilage glycoprotein-39 (HC gp-39), a protein that is differentially expressed at the site of chronic inflammation. Synthetic peptides accommodating the motif were tested for binding the RA-associated DR4 (DRB1*0401) molecules. High-affinity binders were then tested for their capacity to stimulate peripheral blood mononuclear cell responses in RA patients or healthy donors. To assess the arthritogenic nature of native HC gp-39, the protein was injected into BALB/c mice. RESULTS: HC gp-39-derived motif-based peptides were selectively recognized by peripheral blood T cells from RA patients. Injection of the intact protein into BALB/c mice resulted in immunity to HC gp-39, which was found to be associated with the development of a chronic, relapsing arthritis. Moreover, inhalation of the protein led to tolerization of antigen-specific T cells and to suppression of HC gp-39-induced arthritis. CONCLUSION: These data indicate that HC gp-39 is a target of the immune response in RA. Consequently, HC gp-39 is a candidate for antigen-specific immunotherapy.

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The pathogenesis of joint destruction in rheumatoid arthritis remains ill-defined. Joint destruction is thought to be the result of tissue damage mediated by T cells. The mere presence of articular cartilage appears responsible for sustaining chronic synovitis and thereby forwards a role for cartilage-responsive T cells in RA. Taking advantage of the positive DRB1*0401 association with RA susceptibility, we reasoned that T-cell recognition of autoantigens in RA would be restricted by DRB1*0401-encoded molecules. A DR4 (B1*0401) peptide binding motif was used for the identification of putative T-cell epitopes within human aggrecan, a candidate autoantigen. Thirteen peptides were synthesized and tested for binding DRB1*0401 or 0404-encoded molecules. Selected binders were tested for induction of proliferative responses in peripheral blood mononuclear cells from donors carrying the DR4 or DR1 specificity. Both healthy and RA donors responded to human aggrecan-derived peptides, thereby identifying these sequences as T-cell epitopes. Interestingly, responses to aggrecan-derived epitopes were significantly decreased in RA patients compared to controls. This was not due to an overall hyporesponsiveness of RA patients since responses to a recall antigen or mitogen did not differ from controls. The data suggest that in RA, aggrecan-specific T cells may exist in a different stage of activation or may have left the periphery to home to the joint.

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Monoclonal antibodies (mAb) directed against the clonotypic structure of the T-cell receptor (TCR) may be useful reagents in the study and therapy of T-cell-mediated diseases. In contrast to several reports concerning the generation of anti-clonotype mAb to mouse TCR, only very limited numbers of anti-clonotype mAb to human TCR have been described. So far, a suitable method for the generation of anti-clonotype mAb to a given TCR has not been available and in this report we describe a novel strategy for the generation of such mAb. Mice were immunized with intact human T-cells. Then. spleen cell populations were precleared from B-cells reactive to CD3 and the constant region of the TCR by adsorption to TCR/CD3 complexes derived from an irrelevant T-cell clone. Subsequently, clonotype-specific B-cells were selected with TCR/CD3 complexes from the T-cell clone of interest. The small number of B-cells resulting from this selection were clonally expanded in a B-cell culture system and then immortalized by mini-electrofusion. Ten clonotype-specific mAb were generated against a DRB1*0401-restricted T-cell clone recognizing an epitope of the human cartilage glycoprotein 39 (HC gp-39). All mAb immunoprecipitated a heterodimeric 85 kDa protein. Absolute specificity was demonstrated in a T-cell agglutination test with the T-cell clone of interest compared to a set of 16 defined, irrelevant T-cell clones or lines. In functional assays, the mAb were found to inhibit or block antigen-specific T-cell stimulation. In addition, crosslinked mAb were found to stimulate proliferation of the specific clone in the absence of antigen and antigen presenting cells (APC). Such mAb may have clinical relevance in deleting or modulating autoreactive T-cells in a clonotype-specific manner.

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In normal, healthy joints, synovial fibroblasts do not express major histocompatibility complex (MHC) class II molecules. However, in inflamed joints of rheumatoid arthritis (RA) patients, synovial fibroblasts show an abundant expression of MHC class II. Does this increase in expression have functional consequences for antigen presentation to T cells? To date, the precise role of synovial fibroblasts in antigen presentation has not been documented. Here, we show by three different examples that cultured synovial fibroblasts with interferon-gamma (IFN-gamma)-induced MHC class II expression are capable of processing soluble protein for presentation to CD4+ T cells. First, the antigen-presenting cell (APC) function of synovial fibroblasts was studied in an autologous model. From synovial tissue of a RA patient both a fibroblast cell line and a tetanus toxoid (TT)-specific CD4+ T-cell line were generated. A dose-dependent TT response was observed only when TT was presented by IFN-gamma-pretreated synovial fibroblasts. As more direct evidence for MHC class II-restricted antigen presentation, the response of a Mycobacterium tuberculosis-specific CD4+ T-cell clone isolated from rheumatoid synovial fluid was demonstrated in the presence of synovial fibroblasts. The response was DR4Dw4-restricted and could be inhibited by monoclonal antibody (mAb) to HLA-DR. In addition, the lymphokine secretion pattern of the synovial T-cell clone did not differ qualitatively upon antigen-specific stimulation using peripheral blood mononuclear cells (PBMC) or synovial fibroblasts as APC. In order to provide evidence for intracellular antigen processing we next examined the response of a M. leprae-specific T-cell clone with known epitope specificity. Our data suggest that synovial fibroblasts are not passive bystanders, but can become active participants in the development and maintenance of chronic inflammation.

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Prostatic cancer is one of the most frequent forms of malignancy in Western countries. Initially, growth of the majority of prostate tumors can be manipulated by endocrine therapy. However, ultimately androgen independent tumors continue to grow. We studied the expression of oncogenes in four different human prostatic carcinoma cell lines: PC 3, PC 133, PC 135, which are androgen independent, and the hormone dependent PC 82 cell line. Large amounts of Ha-ras and myc mRNA were present in all cell lines. Transcripts of fes, int-1 and abl were never detected. In some of the cell lines the presence of N-ras, Ki-ras, myb, fos, fms and sis mRNA was observed. The PC 82 cell line showed, in addition to myc and Ha-ras high levels of fos expression. Inhibition of tumor cell proliferation by withdrawal of androgen was accompanied by a tenfold reduction of the fos mRNA level and a twofold reduction of Ha-ras transcripts. In contrast, the expression of myc was not changed.

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Initiation of adenovirus DNA replication is dependent on a complex of the precursor of the terminal protein and the adenovirus-coded DNA polymerase (pTP-pol complex). This complex catalyzes the formation of a covalent linkage between dCMP and pTP in the presence of a functional origin of DNA replication residing in the terminal nucleotide sequence of adenovirus DNA. We have purified the pTP-pol complex of adenovirus type 5 and studied its binding to double-stranded DNA. Using DNA-cellulose chromatography it could be shown that the pTP-pol complex has a higher affinity for adenovirus DNA than for calf thymus or pBR322 DNA. From the differential binding of the pTP-pol complex to plasmids containing adenovirus terminal sequences with different deletions, it has been concluded that a sequence of 14 nucleotide pairs at positions 9-22 plays a crucial role in the binding of pTP-pol to adenovirus DNA. This region is conserved in the DNA's of all human adenovirus serotypes and is obviously an important structural element of the adenovirus origin of DNA replication. Comparative binding studies with adenovirus DNA polymerase and pTP-pol indicated that pTP is responsible for the binding. The nature of the binding of pTP-pol to the conserved sequence will be discussed.

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The function of the adenovirus-coded terminal protein and its precursor in viral DNA replication was studied by raising an antiserum against the adenovirus type 5 (Ad5) terminal protein isolated from virions. This antiserum reacted with both the terminal protein and its precursor as measured by a radioimmunoassay. In an in vitro DNA replication system employing nuclear extracts the addition of antiserum inhibits replication when a DNA-terminal protein complex from adenovirions is used as template. The replication of a 3.8% terminal fragment of the Ad2 genome with a protein-free origin (derived from the plasmid XD-7) is also inhibited by the antiserum. This observation confirms a role of the terminal protein precursor in DNA replication. The antiserum completely inhibited the formation of a covalent complex between the precursor terminal protein and dCMP, which is essential for initiation. A function of the terminal protein in the elongation reaction was shown by the inhibitory effect of antiserum on DNA chain elongation in isolated nuclei from Ad5-infected cells. Also in the in vitro DNA replication system employing nuclear extracts the elongation reaction is strongly reduced by addition of the antiserum. These results indicate that the terminal protein and/or its precursor are not only involved in initiation of DNA replication but also in DNA chain elongation.

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The expression of early and intermediate-early viral regions in eight adenovirus type 5 transformed cell lines was analyzed by radioimmuno-inhibition and RNA-DNA hybridization techniques. Details on the arrangement of the integrated viral DNA sequences in these cell lines have already been published (Visser, L., Wassenaar, A.D.C., Van Maarschalkerweerd, M.W. and Rozijn, T.H. (1981) J. Virol, 39, 684-693). In all cell lines tested, proteins encoded by the transforming region E1 are present. Dependent on the viral DNA content, additional early regions are expressed in most cell lines. In two of the cell lines polypeptides related to the adenoviral terminal protein, encoded by the recently described region E2b, could be detected. The viral DNA sequence encoding the body of the terminal protein mRNA is probably integrated intact, but the promoter region and at least some of the leaders are lacking in these cell lines.

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