Defining a T-cell epitope within HSP 65 in recurrent aphthous stomatitis

Abstract

The 65 kD heat shock protein (HSP) has been implicated in the aetiology of recurrent aphthous stomatitis (RAS). We have previously demonstrated that peptide 91-105 derived from the sequence of mycobacterial 65 kD HSP stimulates specifically lymphocytes from patients with RAS. In this investigation, we show that both CD4+ and CD8+ T cells were significantly stimulated with mycobacterial peptide 91-105. In contrast, the human homologous peptide 116-130 stimulated only CD4+ T cells. Inhibition studies showed that CD4+ T cells were class II restricted, whereas CD8+ T cells were class I restricted. We then used truncated or substituted peptides, and demonstrated that residues 95-105 appear to be important, and residue 104(Arg) critical, in stimulating the T cells. Thus, peptide 95- 105 may constitute a T-cell proliferative epitope in RAS. We postulate that the high load of micro-organisms that colonize the oral mucosa may initiate an immune response by the microbial HSP 65-derived peptide 95-105, stimulating the numerous Langerhans cells in the oral mucosa to activate a cross-reacting immune response to the homologous peptide 116-130 within the epithelial HSP 60, initiating the immunopathological changes that lead to RAS.

Fig. 1

Proliferative responses expressed in stimulation indices of enriched CD4 and CD8 cells stimulated with the mycobacterial 65 kD HSP peptide aa 91–105, the homologous human peptide aa 116–130, and control peptide aa 21–35 in patients with RAS; n = 11 for HSP and peptide 91–105, and n = 5 for peptide 116–130 and 21–35. The mean ± s.e.m. disintegrations per minute of cultures without antigen was 1096 (± 191) for CD4 and 919 (± 103) for CD8-enriched cells.

Fig. 2

Inhibition of proliferative responses of PBMC stimulated with HSP 65, using MoAb to HLA class I and class II (n = 9) and TCRγδ (in five patients); isotype controls were MoAb IgG1 and IgG2. (•) Control IgG1; (□) anti-class I (IgG2); (▪) control IgG2; (♦) control IgG1; (○) anti-class II (IgG1); (▵) anti-TCRγδ (IgG1).

Fig. 3

Inhibition of the proliferative responses of PBMC to peptide 91–105 (a) using MoAb recognizing HLA class I and II, and control isotype (n = 9) and (b) TCRγδ (in five patients). (□) Anti-class I (IgG2); (▪) control IgG2; (○) anti-class II (IgG1); (•) control IgG1; (▴) control IgG1; (▵) anti-TCRγδ.

Fig. 4

Inhibition of proliferative responses of PBMC to peptide 116–130 (a) using MoAb to HLA-class I, II (in nine patients) and (b) MoAb to TCRγδ (n = 5). (□) Control IgG1; (•) control IgG2; (○) anti-class I (IgG2); (▪) anti-class II (IgG1); (▵) control IgG1; (▴) anti-TCRγδ.

Fig. 5

Inhibition of enriched (a) CD4 and (b) CD8 cell proliferative responses to peptide 91–105 (n = 4), using MoAb to class II, class I, TCRγδ and the isotype controls at 12·5, 25 and 50 μg/ml. (□) Control IgG1; (○) anti-class I (IgG2); (•) control IgG2; (▪) anti-class II (IgG1); (▵) anti-TCRγδ (IgG1).

Fig. 6

Proliferative responses of PBMC stimulated by peptide 91–105 and truncated peptides (n = 6); dashed line indicates 50% decrease in maximal stimulation with peptide 91–105; (a) shows stimulation indices with N- terminal deletions and (b) with C-terminal deletions.