Binding of peptides of the human acetylcholine receptor alpha-subunit to HLA class II of patients with myasthenia gravis.

MG is an autoimmune disease in which T cells specific to T-cell epitopes of the human acetylcholine receptor play a role. We have identified two peptides, p195-212 and p259-271, of the human acetylcholine receptor alpha-subunit, to which PBLs of MG patients responded by proliferation. Nevertheless, proliferation assays are relatively complicated to perform and might be affected by medications taken by the patients. Therefore, we tested the possibility of using a different assay to determine recognition of these peptides by MG patients. Thus, we performed a direct binding assay using biotinylated peptides and APCs from peripheral blood of MG patients and healthy controls. With this assay we detected the binding of the two peptides to the surface of intact APCs of both MG patients and control donors. Moreover, the presentation of peptide p259-271 by individuals with MG was significantly higher than that observed in healthy subjects. The peptides were specifically bound to HLA class II determinants on the APCs, as shown by inhibition with antibodies to the HLA class II haplotypes of the individuals investigated. Moreover, the binding of these peptides was in correlation with their ability to induce specific proliferative responses of peripheral blood T cells of these patients. The ability to screen for potentially pathogenic epitopes in each patient is of importance for the future design of specific inhibitory analogues that might be used to treat MG.

Measure of transient transfection efficiency using beta-galactosidase protein.

Although the control elements which regulate the transcriptional activity of promoter sequences are largely determined by the use of reporter plasmids in transient transfection analyses, controlling variability in these experiments can often be a vexing problem. Problems arise when the promoter of the internal control plasmid, used to correct for transfection efficiency, either affects test promoter strength or is itself regulated by trans-acting factors or inducing agents used to study the test promoter. Here we report the use of beta-galactosidase protein as an unbiased standard of transfection efficiency. beta-Galactosidase protein is readily internalized by adherent cell lines when incorporated into a calcium phosphate precipitate; significant enzyme activity can be recovered up to 72 h after transfection. Use of beta-galactosidase protein as a control obviates the concerns associated with promoter-dependent reporter plasmids as controls.

Fine specificity of T cell lines and clones that are capable of inducing autoimmune manifestations in mice.

Myasthenia gravis is a T-cell-regulated, antibody-mediated autoimmune disease. The synthetic peptides p195-212 and p259-271, which represent sequences of the human acetylcholine receptor alpha-subunit, preferentially stimulated T cells of patients with myasthenia gravis and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Therefore, we established a p195-212-specific T cell line from SJL mice and a p259-271-specific T cell line from BALB/c mice. N- and C-terminal truncated and/or extended peptides differed in their ability to stimulate proliferative responses of the lines and of their derived clones. Activated cells of the lines were inoculated into naive syngeneic mice. In both strains of mice, peptide-specific antibodies and antibodies to the murine acetylcholine receptor were detected. In addition, decremental compound muscle action potentials consistent with impairment of neuromuscular transmission were recorded from the line-inoculated mice. Thus, these T cell lines, clones, and epitopes constitute a useful model for investigating T cell pathogenicity in autoimmune manifestations related to myasthenia gravis.

Altered peptide ligands of a myasthenogenic epitope as modulators of specific T-cell responses.

Myasthenia gravis (MG) is a T-cell regulated autoimmune disease. A peptide representing a sequence of the human acetylcholine receptor alpha-subunit (p195-212) was previously shown to stimulate proliferative responses of peripheral blood lymphocytes from MG patients and to be an immunodominant and myasthenogenic T-cell epitope in SJL mice. The authors generated a panel of analogues of p195-212 that contain single amino acid substitutions. Three of the analogues (203PHE, 204GLY and 207ALA) triggered low to no proliferative responses of a p195-212-specific T-cell line designated TCSJL195-212. Two of these analogues were able to stimulate the line to produce interleukin-2 (IL-2) and IL-4 (203PHE and 204GLY), whereas one analogue, 207ALA, did not stimulate the line to produce these cytokines. Binding assays revealed that the binding affinity of an altered peptide for a given major histocompatibility complex (MHC) molecule is not sufficient to determine whether it will be stimulatory or inhibitory to a native peptide-specific T-cell line. Two of the analogues, 204GLY and 207ALA, inhibited proliferative responses of cells of the TCSJL195-212 line when co-cultured with p195-212 and syngeneic antigen presenting cells (APC). The two inhibitory analogues were also able to inhibit proliferative responses of the TCSJL195-212 line when APC were pre-pulsed with p195-212, indicating that MHC blockade is not the only mechanism of action of these peptides. Moreover, both analogues inhibited the ability of p195-212 to prime lymph node cells for proliferative responses even when the analogues were administered in a soluble form. Thus the altered peptide ligands 207ALA and 204GLY can modulate T-cell responses both in vitro and in vivo and may have therapeutic potential for the treatment of MG.

T cell receptor expression and differential proliferative responses by T cells specific to a myasthenogenic peptide.

Myasthenia gravis (MG) is a T-cell-regulated autoimmune disease in which a pathological autoantibody response is mounted against the nicotinic acetylcholine receptor of the neuromuscular junction. Our laboratory previously identified a T cell epitope, p195-212, derived from the human acetylcholine receptor alpha subunit, which triggered PBL to proliferate from about 70% of MG patients tested. p195-212 was also found to be an immunodominant T cell epitope in SJL mice and a cryptic epitope in C3H.SW mice. Inoculation of naive SJL mice with cells from a p195-212-specific syngeneic T cell line caused MG-related autoimmune manifestations in those mice. In these studies we analyzed TCR alpha and beta chain sequences used by T cell lines and clones from both high- and low-responder mouse strains in response to p195-212. T cell lines generated from either strain expressed single TCR V beta gene segments (V beta 17 in SJL mice and V beta 8 in C3H.SW mice). By deleting V beta 17-expressing T cells in p195-212-immunized SJL mice we established a T cell line that expressed the V beta 6 gene product, suggesting that SJL mice are not limited to using a single V beta gene segment in response to p195-212. In addition, we found that N- and/or C-terminal-truncated peptides of p195-212, presented under the same culture conditions to different clones bearing the same TCR alpha beta chain, could elicit very different proliferative responses from the clones. Thus, even within a constrained system, factors other than TCR sequence contribute to the differential stimulation of T cell responses.

Inhibition of T-cell reactivity to myasthenogenic epitopes of the human acetylcholine receptor by synthetic analogs.

The synthetic peptides p195-212 and p259-271, representing amino acids 195-212 and 259-271 of the alpha subunit of the human acetylcholine receptor, preferentially stimulate T cells of patients with myasthenia gravis and are immunodominant T-cell epitopes in SJL and BALB/c mice, respectively. We designed and synthesized analogs of these peptides that contain single amino acid substitutions. An analog of peptide p195-212, no. 455 (Met-207-->Ala), was capable of inhibiting up to 100% of the proliferative responses of a p195-212-specific T-cell line originating from the high-responder strain SJL. Similarly, an analog of p259-271, no. 306 (Glu-262-->Lys), was capable of inhibiting up to 93% of the proliferative responses of the p259-271-specific T-cell line originating from high-responder BALB/c mice. Analog 306 also inhibited up to 43% of the proliferative responses of p259-271-primed lymph node cells in an in vitro proliferation assay. To test the in vivo inhibitory activity of the analogs, mice were primed with the myasthenogenic peptides in complete Freund's adjuvant concomitant with administration of the analogs in aqueous solution. Administration of analogs 455 and 306 led to decreased proliferative responses of up to 70% by peptide p199-212-primed lymph node cells and up to 85% by peptide p259-271-primed lymph node cells. Similar results were obtained whether the analogs were administered i.v. or i.p. Thus, these analogs are good candidates for specific immunomodulatory therapy for patients with myasthenia gravis.

Peptide analogs to pathogenic epitopes of the human acetylcholine receptor alpha subunit as potential modulators of myasthenia gravis.

Myasthenia gravis is an autoimmune disease in which T cells specific to epitopes of the autoantigen, the human acetylcholine receptor, play a role. We identified two peptides, p195-212 and p259-271, from the alpha subunit of the receptor, which bound to major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) from peripheral blood lymphocytes of myasthenia gravis patients and stimulated lymphocytes of >80% of the patients. We have prepared analogs of these myasthenogenic peptides and tested their ability to bind to MHC class II determinants and to interfere specifically with T-cell stimulation. We first determined relative binding efficiency of the myasthenogenic peptides and their analogs to APCs of patients. We found that single substituted analogs of p195-212 (Ala-207) and p259-271 (Lys-262) could bind to human MHC molecules on APCs as efficiently as the original peptides. Moreover, dual analogs containing the two single substituted analogs in one stretch (either sequentially, Ala-207/Lys-262, or reciprocally, Lys-262/Ala-207) could also bind to APCs of patients, including those that failed to bind one of the single substituted analogs. The single substituted analogs significantly inhibited T-cell stimulation induced by their respective myasthenogenic peptides in >95% of the patients. The dual analogs were capable of inhibiting stimulation induced by either of the peptides: They inhibited the response to p195-212 and p259-271 in >95% and >90% of the patients, respectively. Thus, the dual analogs are good candidates for inhibition of T-cell responses of myasthenia gravis patients and might have therapeutic potential.

Regulation of major histocompatibility complex class I gene expression in thyroid cells. Role of the cAMP response element-like sequence.

The major histocompatibility complex (MHC) class I gene cAMP response element (CRE)-like site, -107 to -100 base pairs, is a critical component of a previously unrecognized silencer, -127 to -90 bp, important for thyrotropin (TSH)/cAMP-mediated repression in thyrocytes. TSH/cAMP induced-silencer activity is associated with the formation of novel complexes with the 38-base pair silencer, whose appearance requires the CRE and involves ubiquitous and thyroid-specific proteins as follows: the CRE-binding protein, a Y-box protein termed thyrotropin receptor (TSHR) suppressor element protein-1 (TSEP-1); thyroid transcription factor-1 (TTF-1); and Pax-8. TTF-1 is an enhancer of class I promoter activity; Pax-8 and TSEP-1 are suppressors. TSH/cAMP decreases TTF-1 complex formation with the silencer, thereby decreasing maximal class I expression; TSH/cAMP enhance TSEP-1 and Pax-8 complex formation in association with their repressive actions. Oligonucleotides that bind TSEP-1, not Pax-8, prevent formation of the TSH/cAMP-induced complexes associated with TSH-induced class I suppression, i.e. TSEP-1 appears to be the dominant repressor factor associated with TSH/cAMP-decreased class I activity and formation of the novel complexes. TSEP-1, TTF-1, and/or Pax-8 are involved in TSH/cAMP-induced negative regulation of the TSH receptor gene in thyrocytes, suppression of MHC class II, and up-regulation of thyroglobulin. TSH/cAMP coordinate regulation of common transcription factors may, therefore, be the basis for self-tolerance and the absence of autoimmunity in the face of TSHR-mediated increases in gene products that are important for thyroid growth and function but are able to act as autoantigens.