Discrepant Expression Between mRNA and Protein of HTLV-I Basic Leucine Zipper Factor (HBZ) Makes HTLV-I Infected CD4 Lymphocytes and Adult T Cell Leukemia Cells Less Susceptible to HBZ-Specific Cytotoxic T Lymphocytes

Abstract

[Background]Adult T cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and still remains one of the chemotherapy-resistant leukemias. Accumulating the successful evidence of allogeneic hematopoietic stem cell transplantation (allo-HSCT), the immunotherapeutic strategy for ATL has become to look promising. The fact that more than half of primary ATL cells lack the immunogenic oncoprotien HTLV-1 Tax has been promoting researchers to find an alternative target antigen to develop the cellular immunotherapy for ATL. Recently, HTLV-1 basic leucine zipper factor (HBZ), which is encoded by the minus strand of HTLV-1 proviral genome and is transcribed from 3'-LTR, has been highlighted as the important molecule in ATL leukemogenesis. Since almost all ATL cells express HBZ mRNA, we attempted to verify whether HBZ can be a target of cellular immunotherapy for ATL.[Methods] At first, we synthesized a variety of HBZ-derived 9 amino acid peptides (9 mer) that are predicted to have high binding affinity to HLA-A*0201 molecule. CD8+ T lymphocytes from an HLA-A*0201+ healthy donor were stimulated with peptide-loaded autologous monocyte-derived mature dendritic cells repetitively. Thereafter, epitope specificity, HLA-restriction, and cytotoxic activity of induced cytotoxic T lymphocytes (CTLs) were determined by standard 51Cr-release assays. HBZ and Tax mRNA expression levels of target cells, including primary ATL cells, HTLV-1-infected cell lines, peripheral blood lymphocytes isolated from HTLV-1 carriers and HTLV-1-uninfected individuals, and K562-A*0201 and C1R-A*0201 cells transfected with HBZ gene, were simultaneously measured by real-time quantitative PCR (RQ-PCR). The relative expression levels of HBZ and Tax mRNA were determined by comparative Ct method, and the results were shown as the relative values to those of HTLV-1-infected cell line MT4. HBZ protein expression was determined by Western blotting using anti-HBZ serum which was produced by immunizing rabbits with purified six-His-tagged HBZ polypeptide corresponding to the bZIP domain of HBZ. The detection of HBZ-specific and Tax-specific CTLs was performed by tetramer assays.[Results] We identified an HBZ-derived 9-mer epitope, HBZ26–34 (GLLSLEEEL), with high binding affinity to HLA-A*0201 measured by using HLA-A*0201 gene-transfected T2 cell. We successfully established an HBZ26–34 peptide-specific CTL clone designated as HBZ-1. HBZ-1 exerted cytotoxicity against HBZ gene-transfected K562-A*0201 and C1R-A*0201 cells, but not against HLA-A*0201-positive HTLV-1-infected cells or ATL cells. HTLV-1 infection and HBZ gene transfection did not alter the HLA class I expression of target cells. Expression levels of HBZ mRNA appeared to be higher in ATL cells, HTLV-1-infected cell lines, and HBZ gene-transfected cells than those in HTLV-1 carrier cells. Abundant HBZ protein expression was detected in only HBZ gene-transfected K562-A*0201 and C1R-A*0201 cells, but HBZ protein expression levels in HTLV-1-infected cell lines and ATL cells appeared to be very low. We further examined Tax-specific and HBZ-specific CTLs in an HLA-A*0201-positive ATL patient who received allo-HSCT from the HLA-identical sibling donor. In this patient, Tax mRNA and HBZ mRNA were similarly expressed in ATL cells; however, only Tax-specific CTLs but not HBZ-specific CTLs were detected in both before and after allo-HSCT at full donor chimerism.[Conclusions] Although HBZ mRNA was apparently detected in all HTLV-1-infected cell lines and ATL cells, HBZ protein in these cells was insufficiently expressed to be recognized by HBZ-specific CTLs. Our observations in a transplanted patient with ATL also suggest that HBZ protein may be less immunogenic due to discrepant expression level between mRNA and protein.