Scanning a DRB3*0101 (DR52a)-restricted epitope cross-presented by DR3: overlapping natural and artificial determinants in the human acetylcholine receptor.

A recurring epitope in the human acetylcholine receptor (AChR) alpha subunit (alpha146-160) is presented to specific T cells from myasthenia gravis patients by HLA-DRB3*0101-"DR52a"-or by DR4. Here we first map residues critical for DR52a in this epitope by serial Ala substitution. For two somewhat similar T cells, this confirms the recently deduced importance of hydrophobic "anchor" residues at peptide p1 and p9; also of Asp at p4, which complements this allele's distinctive Arg74 in DRbeta. Surprisingly, despite the 9 sequence differences in DRbeta between DR52a and DR3, merely reducing the bulk of the peptide's p1 anchor residue (Trp149-->Phe) allowed maximal cross-presentation to both T cells by DR3 (which has Val86 instead of Gly). The shared K71G73R74N77 motif in the alpha helices of DR52a and DR3 thus outweighs the five differences in the floor of the peptide-binding groove. A second issue is that T cells selected in vitro with synthetic AChR peptides rarely respond to longer Ag preparations, whereas those raised with recombinant subunits consistently recognize epitopes processed naturally even from whole AChR. Here we compared one T cell of each kind, which both respond to many overlapping alpha140-160 region peptides (in proliferation assays). Even though both use Vbeta2 to recognize peptides bound to the same HLA-DR52a in the same register, the peptide-selected line nevertheless proved to depend on a recurring synthetic artifact-a widely underestimated problem. Unlike these contaminant-responsive T cells, those that are truly specific for natural AChR epitopes appear less heterogeneous and therefore more suitable targets for selective immunotherapy.

A pathogenetic role for the thymoma in myasthenia gravis. Autosensitization of IL-4- producing T cell clones recognizing extracellular acetylcholine receptor epitopes presented by minority class II isotypes.

Myasthenia gravis (MG) is caused by helper T cell-dependent autoantibodies against the muscle acetylcholine receptor (AChR). Thymic epithelial tumors (thymomas) occur in 10% of MG patients, but their autoimmunizing potential is unclear. They express mRNAs encoding AChR alpha and epsilon subunits, and might aberrantly select or sensitize developing thymocytes or recirculating peripheral T cells against AChR epitopes. Alternatively, there could be defective self-tolerance induction in the abundant maturing thymocytes that they usually generate. For the first time, we have isolated and characterized AChR-specific T cell clones from two MG thymomas. They recognize extracellular epitopes (alpha75-90 and alpha149-158) which are processed very efficiently from muscle AChR. Both clones express CD4 and CD8alpha, and have a Th-0 cytokine profile, producing IL-4 as well as IFN-gamma. They are restricted to HLA-DP14 and DR52a; expression of these minority isotypes was strong on professional antigen-presenting cells in the donors' tumors, although it is generally weak in the periphery. The two clones' T cell receptor beta chains are different, but their alpha chain sequences are very similar. These resemblances, and the striking contrasts with T cells previously cloned from non-thymoma patients, show that thymomas generate and actively induce specific T cells rather than merely failing to tolerize them against self antigens.

Epitopes expressed in myasthenia gravis (MG) thymomas are not recognized by patients' T cells or autoantibodies.

Most thymic epithelial tumours that associate with MG express an epitope that resembles the sequence alpha373-380 from the cytoplasmic loop of the acetylcholine receptor (AChR). It has been proposed that sensitization to this linear epitope initiates autoimmunity to the AChR in thymoma-associated MG. We therefore tested whether MG/thymoma patients have T cell responses or antibodies to this region of the AChR. We found no significant recognition of the alpha309-417 region by their thymoma or peripheral blood T cells, or by their serum anti-AChR antibodies. Instead, the T cell epitopes that were recognized, like the previously characterized B cell epitopes, were in the extracellular AChR domain.

Oncogene proteins and proliferation antigens in thymomas: increased expression of epidermal growth factor receptor and Ki67 antigen.

AIMS: To examine thymomas for proteins encoded by oncogenes and to determine whether their presence correlates with tumour growth and associated myasthenia gravis. METHODS: Sections of 24 thymomas were incubated with anti-EGF receptor (EGF-R), anti-Ki67 antigen, anti-p53, and anti-bcl-2 antibodies, and then stained using the alkaline phosphatase/anti-alkaline phosphatase (APAAP) technique. Cell suspensions and epithelial cell cultures from some of the tumours were also studied. RESULTS: Whereas EGF-R expression was not detected in any of the controls (but only in a 20 week old fetus), it was detected in neoplastic epithelial cells of all thymomas, and was most strongly expressed in metastases and in samples from donors with severe myasthenia gravis. Ki67 labelling was also increased, especially in the larger thymomas. Epithelial expression of both of these markers was confirmed in fresh cell suspensions and monolayer cultures from the five available cases. In contrast, p53 and bcl-2 were not detected in the neoplastic cells, but bcl-2 was present in the intermingling thymocytes. CONCLUSIONS: Neoplastic thymoma cells express EGF-R and Ki67, but there is no concomitant increase in the expression of p53 and bcl-2 proteins. Increased EGF-R expression may result in increased proliferation of neoplastic cells and also in myasthenia gravis. Measurement of EGF-R concentrations may be of prognostic value. The bcl-2 staining pattern in T lymphocytes illustrates the broad spectrum of maturational stages in thymoma lymphocytes.

Antigen presentation by thymoma epithelial cells from myasthenia gravis patients to potentially pathogenic T cells.

Thymomas associate strongly with myasthenia gravis (MG). We now show that cultured thymoma epithelial cells can present synthetic acetylcholine receptor (AChR) peptides to HLA-sharing responder T cell lines/clones nearly as efficiently as blood mononuclear cells. Responses depended strictly on the specific antigen added. Processing of longer recombinant AChR polypeptides was clearly less efficient than by blood mononuclear cells, and was selectively abolished by preculture with chloroquine. The T cell responses depended on the presence of LFA-3 on the thymoma cells. This study demonstrates that thymoma epithelial cells have the capacity to stimulate T cells and perhaps, therefore, to autosensitize against AChR in vivo.

Approaches for studying the pathogenic T cells in autoimmune patients.

Our provisional conclusions from this work are as follows. (1) For screening responses of established lines, native human AChR is not prohibitively scarce, especially if it is concentrated onto beads, and class II-transfected TE671 cells may be useful too; both may give vital evidence of AChR-specificity, but it is still crucial to confirm that with synthetic peptides. (2) For mapping epitopes, panels of full-length and shorter recombinant human polypeptides, and of synthetic peptides, are invaluable complementary material: longer peptides tend to stimulate particularly strongly. (3) Initial selection with pooled synthetic peptides can easily generate interesting lines from both patients and controls, but they may depend on the artificial processing sites that are an inevitable consequence of arbitrarily chosen start and stop points. Of course, these might conceivably be employed in unusual antigen-presenting cells (such as thymic myoid cells), so we cannot totally dismiss such "cryptic" epitopes. This system can sometimes select T cells responding to "natural" epitopes too, as now reported for tetanus toxin. Nevertheless, for these and other reasons, at present, we strongly favor using the longest human recombinant material possible, because it is apparently processed more naturally. This must be combined with rigorous screening for reactivity to E. coli-derived contaminants plus concomitant mapping of epitopes as above. Use of intact AChR for initiating lines may yet become feasible. (4) The T cells thus isolated and characterized so far are proving to be heterogeneous in the epitopes and presenting class II molecules they recognize, and in their T-cell receptor gene usage. It is premature to claim key myasthenogenic epitopes or clonotypes, but HLA-DR3 and the linked -DQw2 do not appear to monopolize presentation. (5) Assessing the disease-relevance of these T cells is a separate problem, highlighted by their apparent similarity in healthy controls. In the meantime, to test their potential pathogenicity, we are assaying their cytokine profiles and ability to help specific antibody production in vitro. In the hope that they do prove to be relevant, we are also using some of them to test possible therapeutic strategies that might prove applicable in the patients.