No abstract

A crude supernatant of hybridoma secreting a monoclonal anti-double-stranded (ds)DNA antibody (PME77 mAb), used to stain fibroblasts (CVI cells) in immunofluorescence, gives a punctuated staining of variable intensity. We had suggested that anti-DNA antibodies bind to cell-surface protein(s) of several cells. When the mAb of this crude supernatant was purified on a dsDNA-cellulose column and a histoneTrisacryl column, the mAb no longer bound to the cell surface. Only when dsDNA plus purified histones was added to the purified antibody did the immune complex strongly and uniformly stain again the cell surface of CVI cells. No significant staining was observed if either DNA or histones were omitted. A single 94-kDa protein from membrane fractions ofCVI, Raji, and RINm cell lines was visualized in immunoblots when mAb-DNA-histone complexes were applied to the nitrocellulose strips. No polypeptide was seen if one component was omitted. This 94-kDa protein behaved like a plasma membrane protein since it required the use of detergent to be solubilized and was quantitatively recovered in the Triton X-114 detergent-rich phase. Moreover, a brief treatment of living cells with trypsin cleared off this protein. Purified nucleosomes could be substituted to DNA-histone complexes, giving rise to identical results. Finally, purified polyclonal anti-DNA antibodies from sera of systemic lupus erythematosus patients labeled a 94-kDa protein provided that DNA-histone complexes were added. Anti-DNA autoantibodies could be pathogenic when they are bound to nucleosomes.

INSERM U 25, HBpital Necker, ParisXeno-sensitization of human peripheral blood lymphocytes (PBL) against mouse lymphoid cells has been studied in vivo in a local graft-vs. -host (GVH) assay and in virro in a mixed lymphocyte culture-cell-mediated lympholysis system. Human PBL were injected into the footpads of mice rendered unresponsive by total-body irradiation, and these were subsequently tested for the presence of cytotoxic cells in the draining popliteal lymph node (LN). In spite of a definite PBL-induced LN proliferation, n o cytotoxic activity was detected against mouse target cells. In contrast with the GVH assay, human PBL collected after 7 days of in vitro culture in the presence of irradiated mouse cells, were strongly cytotoxic against mouse target cells. The antigenic specificities recogniied by in vitro educated cells were primarily those coded for by the H-2 complex. Only target cells with a n H-2 haplotype identical to that of the sensitizing mouse strain or with a t least an H-2D end in common with that strain were killed by xeno-sensitized lymphocytes. Mouse target cells derived from congenic resistant strains remained unaffected. It was verified that in vitro educated PBL depleted of B cells retained their cytotoxic effect, indicating that non-B cells and probably T cells were involved in the response .

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease with a predominantly non-hereditary etiology that results in a destruction of pancreatic beta cells by autoaggressive T lymphocytes. Neither the mechanism of initial stimulation of these T cells nor the nature of the environmental factors implicated in the disease have so far been identified. However, both issues are taken into account by the hypothesis of initial T cell activation by viral or bacterial mimicry peptides with sequence similarities to pancreatic self antigens. We determined sequential epitope motifs to search for mimicry peptides stimulating T cell lines specific for two epitopes derived from the IDDM autoantigen 65-kDa glutamic acid decarboxylase (GAD65). These were GAD65 (88-99), presented by HLA-DRB1*0101, and GAD65 (248-257), presented by HLA-DRB5*0101. T cell stimulation by peptides with substitutions in HLA anchor or T cell contact positions was analyzed to establish degenerate epitope motifs for database searching. Out of 28 tested candidate mimicry peptides derived from bacterial, viral and human proteins, 3 stimulated T cell lines and a T cell clone specific for epitope GAD65 (248-257). Our results demonstrate that mono- and polyclonal GAD65-specific T cells from IDDM patients can be stimulated by viral and bacterial peptides with little apparent sequence homology with autoantigenic epitopes. Moreover, in a synopsis with related published studies, our findings suggest that simple degenerate search motifs comprising principal T cell contacts plus HLA class II binding motifs may suffice to identify most mimicry peptides.