Peptides from NM23B - A Transcription Factor with NDP Kinase Activity - Are Expressed on the Surface of Leukemic Cells and Are Recognized by T-Lymphocytes.

Objective: During the last years a growing number of MHC-restricted antigens were recognized using autologous or HLA-matched cytotoxic T-cell lines (TCL). Molecules were isolated by HPLC or identified using cDNA expression cloning from normal or malignant target cells and found to derive from normal proteins or from mutated tumor-specific proteins. The majority of the tumor-specific peptides were derived from melanoma cells. The aim of this project was to search for immunogenic peptides on leukemia cells with the help of TCLs obtained from a stem cell donor against chronic myelogenous leukaemia (CML) recipient cells and to identify the immunogenic peptides by cDNA expression cloning.

Methods: Irradiated leukemic cells of a patient with Philadelphia-chromosome positive CML were used to stimulate PBMC of his HLA-identical related donor. TCLs were expanded to >200x10⁶ cells with antigen specific stimulation and their specificity tested in in vitro assays. Restriction of the TCLs was tested in an Elispot assay using a panel of normal and malignant autologous and allogeneic cells and mAb blocking experiments (mAb W6.32, B1.23.2, SRF8-B6, GAP-A3). Subsequently cDNA libraries from leukemic cells were established in the eukaryontic expression vector pcDNA3.1/V5HISTOPO and cDNA of 100 bacterial colonies were pooled. 293T cells were then co-transfected with the cloned HLA-cDNA of the restricting HLA and with pools of cDNA (pools of 100) obtained from the leukemic cells. Transfected cells were tested with the TCL in the Elispot assay. Positive cDNA pools were further diluted in pools of 10 colonies and tested again. Positive pools were finally diluted in single clones and screened again. cDNA from the positive single clones was sequenced with different primers.

Results: The TCL PaHe recognized bcr/abl positive PBMC, CD14⁺ and CD34⁺ subpopulations and EBV-LCL but not autologous CD4⁺ and CD8⁺ subpopulations or bcr/abl negative PHA blasts. Blocking experiments showed that the recognized peptides were restricted by HLA-A32, HLA-B44 and HLA-C03. Therefore 293T cells were transfected with HLA-A32 and with the cDNA pools of the tumor cells. After testing one thousand 100-cDNA pools and four hundred 10 cDNA pools a total of three single cDNA clones were identified, which after transfection were recognized by the TCL. All three positive cDNAs clones were sequenced and found to be identical with the sequence of NM23B. No mutations of the molecule were detected even if different primers were used.

Conclusions: Peptides from NM23B, a transcription factor for c-myc with nucleosid diphosphate kinase (NDP) activity, are recognized by TCLs in an HLA-A32 restricted way. This is the first report of NM23B as an immunogenic molecule, which might play an important role in graft-versus-tumor reactions but also in the regulation of hematopoiesis.