HLA-A2 restricted, melanocyte-specific CD8(+) T lymphocytes detected in vitiligo patients are related to disease activity and are predominantly directed against MelanA/MART1.

Vitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions due to lack of melanocytes in the respective areas. It has been suggested that vitiligo is caused by an autoimmune-mediated destruction of melanocytes. Recently, the presence of a high frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in the peripheral blood of patients with vitiligo was reported. Our study examines the frequency of melanocyte-specific cytotoxic T lymphocytes in vitiligo patients and its relationship to disease activity. Thirty-two patients with moderate to active vitiligo and 17 control subjects were included. Melanocyte specific reactive CD8(+) T cells were identified by enzyme-linked immunospot assay after stimulation with five peptides from gp100, four peptides from MelanA/MART1, and two peptides from tyrosinase. In selected patients, intracellular interferon-gamma staining for the detection of specific reactive CD8(+) T cells was additionally performed. In seven of 10 patients (70%) with actively progressive disease CD8(+) T cells directed against melanocyte epitopes were detected, whereas only in four of 22 patients (18%) with moderate disease activity such specific reactivity was found. MelanA/MART1 peptides were immunodominant in nine patients reacting against EAAGIGILTV and three patients reacting against ILTVILGVL. Intracellular interferon-gamma staining confirmed the findings obtained by the enzyme-linked immunospot technique. The present study supports the hypothesis that vitiligo is a cytotoxic T lymphocyte-mediated autoimmune disease. The presence of melanocyte-specific reactive CD8(+) T cells seems to be closely related to disease activity.

Ex vivo generation of human cytomegalovirus-specific cytotoxic T cells by peptide-pulsed dendritic cells.

Adoptive transfer of donor-derived human cytomegalovirus (HCMV)-specific T-cell clones can restore protective immunity after stem cell transplantation. Ex vivo induction of HCMV-specific T cells using HCMV-infected fibroblasts as stimulator cells confines this approach to HCMV-seropositive donors and requires the presence of infectious virus during the stimulation procedure. In this study, we describe a potential alternative strategy to generate HCMV-specific T cells ex vivo for adoptive immunotherapy. Generation of HCMV-specific cytotoxic T lymphocytes (CTLs) ex vivo was investigated using peptide-pulsed dendritic cells as antigen-presenting cells. HCMV-specific T cells were generated and sufficiently expanded for adoptive immunotherapy in 6 out of 14 HCMV-seropositive and 2 out of 11 HCMV-seronegative donors. The CTLs recognized HCMV-infected autologous fibroblasts. No lysis was observed with either non-infected autologous or HLA-mismatched infected fibroblasts. Staining with tetrameric HLA/peptide complexes revealed significant enrichment for peptide-specific T cells of up to 28% and > 90% of CD8(+) T cells after three and five specific stimulations respectively. In addition, the expansion rates indicated that ex vivo generation of > 1 x 10(9) HCMV-specific T cells was possible after 6--7 weeks when cultures were initiated with 1--5 x 10(6) responder cells. Thus, the approach with peptide-pulsed DCs to generate HCMV-specific CTLs is feasible for clinical application after allogeneic stem cell transplantation.

Identification of HLA-A2-restricted T-cell epitopes derived from the MUC1 tumor antigen for broadly applicable vaccine therapies.

The tumor-associated antigen MUC1 is overexpressed on various hematological and epithelial malignancies and is therefore a suitable candidate for broadly applicable vaccine therapies. It was demonstrated that major histocompatibility complex (MHC)-unrestricted cytotoxic T cells can recognize epitopes of the MUC1 protein core localized in the tandem repeat domain. There is increasing evidence now that MHC-restricted T cells can also be induced after immunization with the MUC1 protein or segments of the core tandem repeat. Using a computer analysis of the MUC1 amino acid sequence, we identified two novel peptides with a high binding probability to the HLA-A2 molecule. One of the peptides is derived from the tandem repeat region and the other is derived from the leader sequence of the MUC1 protein, suggesting that, in contrast to previous reports, the MUC1-directed immune responses are not limited to the extracellular tandem repeat domain. Cytotoxic T cells (CTL) were generated from several healthy donors by primary in vitro immunization using peptide-pulsed dendritic cells. The addition of a Pan-HLA-DR binding peptide PADRE as a T-helper epitope during the in vitro priming resulted in an increased cytotoxic activity of the MUC1-specific CTL and a higher production of cytokines such as interleukin-12 and interferon-gamma in the cell cultures, demonstrating the importance of CD4 cells for an efficient CTL priming. The peptide induced CTL lysed tumors endogenously expressing MUC1 in an antigen-specific and HLA-A2-restricted fashion, including breast and pancreatic tumor cells as well as renal cell carcinoma cells, showing that these peptides are shared among many tumors. The use of MUC1-derived peptides could provide a broadly applicable approach for the development of dendritic cell-based vaccination therapies.