High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo

Abstract

Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. Using tetrameric complexes of human histocompatibility leukocyte antigen (HLA) class I to identify antigen-specific T cells ex vivo, we observed high frequencies of circulating MelanA-specific, A*0201-restricted cytotoxic T lymphocytes (A2-MelanA tetramer+ CTLs) in seven of nine HLA-A*0201-positive individuals with vitiligo. Isolated A2-MelanA tetramer+ CTLs were able to lyse A*0201-matched melanoma cells in vitro and their frequency ex vivo correlated with extent of disease. In contrast, no A2-MelanA tetramer+ CTL could be identified ex vivo in all four A*0201-negative vitiligo patients or five of six A*0201-positive asymptomatic controls. Finally, we observed that the A2-MelanA tetramer+ CTLs isolated from vitiligo patients expressed high levels of the skin homing receptor, cutaneous lymphocyte-associated antigen, which was absent from the CTLs seen in the single A*0201-positive normal control. These data are consistent with a role of skin-homing autoreactive melanocyte-specific CTLs in causing the destruction of melanocytes seen in autoimmune vitiligo. Lack of homing receptors on the surface of autoreactive CTLs could be a mechanism to control peripheral tolerance in vivo.

Figure 1

Uncultured CD8⁺ T cells stained with A2–MelanA tetramer and anti–CLA antigen from: (A) an A*0201-positive patient with vitiligo in whom the majority of MelanA-specific CTLs are positive for CLA (percentages given in each quadrant); (B) an A*0201-positive normal control individual in whom there were no detectable MelanA-specific CTLs ex vivo (pattern observed in five of six normal controls); (C) the A*0201- positive normal control individual in whom there were detectable MelanA-specific CTLs ex vivo, but which were all negative for CLA (pattern observed in one of six normal controls); (D) an A*0201-negative patient with vitiligo in whom there were no detectable A2–MelanA tetramer⁺ CTLs (observed in all A*0201-negative vitiligo patients). E shows the percentage of CD8⁺ T cells staining with A2–MelanA tetramer in the A*0201-positive normal controls and A*0201-positive vitiligo patients. F confirms a significant difference between the percentage of CLA-positive MelanA-specific CTLs in the PBMC of A*0201-positive patients and controls (P < 0.05).

Figure 2

Optimized peptide-stimulated IL-7–based cultures showing the CD8⁺ T cells stained with A2–MelanA tetramer and anti–CLA from: (A) an A*0201-positive patient with vitiligo in whom the majority of cultured MelanA-specific CTLs are positive for CLA (percentages given in each quadrant); (B) an A*0201-positive normal control individual in whom there was a low frequency of cultured MelanA-specific CTLs that were predominantly negative for CLA (pattern observed in five of six normal controls); (C) the A*0201-positive normal control individual in whom there were detectable MelanA-specific CTLs ex vivo, and in whom it was possible to culture high levels of MelanA-specific CTLs that were all persistently negative for CLA (pattern observed in one of six normal controls); (D) an A*0201-negative patient with vitiligo in whom there were no detectable A2–MelanA tetramer⁺ CTLs (observed in all A*0201-negative vitiligo patients) after a period of stimulation in vitro. E shows the percentage of CD8⁺ T cells from the cultures staining with A2–MelanA tetramer in the A*0201-positive normal controls and A*0201-positive vitiligo patients. F shows a significant difference between the percentage of CLA-positive MelanA-specific CTLs in the cultures from A*0201-positive patients and controls (P < 0.05).

Figure 3

(A–C) Cytolytic activity of MelanA/A*0201-specific CTL lines derived from an A*0201-positive vitiligo patient either unsorted (A), enriched for CD8⁺ A2–MelanA tetramer⁺ cells (B) or enriched for CD8⁺ A2–MelanA tetramer⁻ cells (C). The unsorted and CD8⁺-tetramer⁺ cells were both able to lyse peptide-pulsed targets, but only the CD8⁺ A2–MelanA tetramer⁺ population showed significant lysis of A*0201-matched melanoma cells (ME 275 and SK-mel-29). (D–E) The corresponding percentages of CD8⁺ T cells staining with the A2–MelanA tetramer are shown: the unsorted population contained 17% antigen-specific cells (D); the CD8⁺ A2–MelanA tetramer⁺ population contained 72% antigen-specific cells (E); and the CD8⁺ A2–MelanA tetramer⁻ population contained 1.5% antigen-specific cells.