Comprehensive Analysis of MHC Class I Peptides Presented on Adult T Cell Leukemia Cells by Mass-Spectrometry.

[Objective]Adult T cell leukemia (ATL) is extremely resistant to conventional chemotherapy and hard to cure even with allogeneic stem cell transplantation. Cytotoxic T-lymphocyte-based immunotherapy is expected to be one the therapeutic options for ATL in the near future. So far, however, there are no promising leukemia-associated antigens for immunotherapy of ATL. It is known that most leukemic cells of ATL in vivo hardly express HTLV-I-associated viral antigens. Furthermore, in contrast to solid tumors, only a few tumor (leukemia)-associated antigens have been identified in hematological malignancies. Thus, it is crucial for establishment of immunotherapy of ATL to identify novel applicable leukemia-associated antigens. In the present study, we attempted a comprehensive analysis of MHC class I peptides presented on ATL cells by the latest technology of mass-spectrometry (MS) which has recently made rapid progress.

[Materials&Methods]Three ATL-derived cell lines, ED-D40515, ATL-43T, and ATL-55T+ were cultured and expanded on a large scale, each of which has been demonstrated to be clonally identical to original leukemic cells by southern blot analysis of either HTLV-I provirus or TCR rearangement. Cell lysates prepared from 2–3×10⁹ cells were incubated with Affigel-Hz (Bio-Rad) coupled with anti-HLA class I mAb (W6/32). Affigel resin beads were washed extensively and then subjected to elution with acidic buffer. MS analyses were performed using a LCQ Deca ion trap instrument (ThermoQuest) equipped with a directly communicated on-line reversed- phase high performance liquid chromatography (RP-HPLC) system (MichromBioResources). Protein identification was performed using the SEQUEST algorithm maintained at the National Center for Biotechnology Information (NCBI). We analyzed MHC class I restriction of the candidate peptides and assorted them based on the algorithm of HLA ligands databases SYFPEITHI.

[Results]We were able to sequence approximately 60 peptides per cell line which were derived from different source proteins and presented by respective HLA class I alleles. Although most peptides were derived from ubiquitous or constitutively expressed proteins in normal CD4+ T cells, 10 source proteins that we identified had been previously reported as the cancer-testis antigens or the cancer-associated and/or -overexpressed gene products. We are currently screening most peptides including those derived from unknown gene products by comparing mRNA expression in ATL cells and normal tissues. Up to now, 7 candidate peptides have been identified that seem to be derived from leukemia-associated antigens specifically expressed in ATL cells.

[Discussion]Recent progress in proteomics technology has enabled us to directly determine sequences of numerous peptides bound to MHC molecules. In the present study, we demonstrated that LC-MS is a feasible and efficient method to analyze comprehensive repertoire of MHC class I peptides presented on ATL cells. Several candidate peptides presumably derived from leukemia-associated or -overexpressed antigens were identified based on MHC class I restriction and mRNA expression pattern in leukemic cells and normal tissues. It is to be tested whether these peptides can induce bonafide CTL response against ATL cells and at the end be applicable to immunotherapy of ATL. Considering that expression patterns of leukemia-associated antigens vary from case to case, this approach appears to be suitable for the tailor-made immunotherapy.