Spontaneous CD8 T cell responses against the melanocyte differentiation antigen RAB38/NY-MEL-1 in melanoma patients.

The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-1(50-58) exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 T cells from HLA-A *0201(+) melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of natural processing of RAB38/NY-MEL-1(50-58) peptide. Accordingly, monoclonal RAB38/NY-MEL-1(50-58)-specific T cell populations were capable of specifically recognizing HLA-A2(+) melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2/RAB38/NY-MEL-1(50-58) multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY-MEL-1(50-58) is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials.

Telomere maintenance in human B lymphocytes.

Telomere shortening has been causally linked to replicative senescence in human cells. To characterize telomere-length heterogeneity in peripheral blood cells of normal individuals, we analysed the mean length of telomeric repeat sequences in subpopulations of peripheral blood leucocytes, using fluorescence in situ hybridization and flow cytometry (flow-FISH). Although the telomere length of most haematopoietic subsets was within the same range, the mean telomere length was found to be 15% higher in B compared with T lymphocytes in adult peripheral blood. Whereas telomere loss with ageing corresponded to 33 base pairs (bp) per year in T cells, telomere shortening was slower in B cells, corresponding to 15 bp per year. Separation of adult B-lymphocyte subpopulations based on CD27 expression revealed that telomere length was almost 2 kb longer in CD19+CD27+ (memory) compared with CD19+CD27- (naive) cells. Furthermore, peripheral blood B cells were activated in vitro. Whereas B-cell activation with Staphylococcus aureus Cowan strain (SAC) did not increase telomere length, a striking telomere elongation was observed when cells were stimulated with SAC and interleukin 2 to induce plasma cell differentiation. Our observations support the concept that telomere dynamics in B cells are distinct from other haematopoietic cell lineages and that telomere elongation may play an essential role in the generation of long-term B memory cells.