Identification And Characterization Of P210^bcr-abl-restricted T Lymphocyte Epitopes In Chronic Myeloid Leukemia

The BCR-ABL chimeric oncogene is caused by reciprocal translocation between long arms of chrosome 9 and 22. It encodes a hybrid protein, called p210^bcr-abl, which mediating constitutive tyrosine kinase activity. The protein of p210^bcr-abl is not only associated with malignant transformation of myeloid hematopoietic stem cells or progenitors, but can elicit immune response. As a tumor specific antigen in chronic myeloid leukemia (CML), how it induces cellular and humoral immunity now still remains elusive. Our group had predicted B cell epitopes of 151 amino acids around BCR-ABL fusion region. In this study, we identified two T cell epitopes of p210^bcr-abl and analyzed their biologic features in chronic myeloid leukemia patients.Our study is divided into three parts.Part I: Prediction and Identification of HLA-A*0201-restricted T cell epitopes from p210^bcr-ablAs HLA-A * 0201 is the most common allele in Han population, we preferred to predict HLA-A * 0201-restricted T cell epitopes of p210^bcr-abl. At first, T cell epitopes of full length and fusion region in p210^bcr-abl were analyzed by two algorithms (BIMAS and SYFPEITHI), respectively. Then the degradation by proteasome was analyzed with a software of NetChop3.0. Based on the prediction results by three algorithms, we selected one epitope in the fusion region (SSKALQRPV) and another epitope (LLYKPVDRV) outside the fusion region. Each epitope got the highest score in its region.Due to low affinity between SSKALQRPV and HLA-A*0201, the epitope of SSKALQRPV was mutated to YLKALQRPV, which would greatly enhance the affinity. Lastly we assessed the binding of the epitopes using a MHC stabilization assay on live T2 cells. The results showed that two epitopes, LLYKPVDRV (named p210^bcr-abl642) and YLKALQRPV (named p210^bcr-abl926m), had high affinity with HLA-A*0201 molecules.Part II: Characterization of epitope-restrictive CTLs in CML patientsIn order to select HLA-A*0201-positive individuals, mononuclear cells were isolated with Lymphocyte Separation Media from bone marrow or peripheral blood, next stained with HLA-A*0201 monoclonal antibody and analyzed by flow cytometry. In total, there were 6 HLA-A*0201-positive healthy donors and 14 HLA-A*0201-positive CML patients. In the assay of detecting epitope-specific CTLs, lymphocytes were labeled with soluble HLA-A*0201 tetramer and CD8 antibody. Data of double positive cells were collected on a FACSCalibur flow cytometer. The results showed that percentages of p210^bcr-abl642/p210^bcr-abl926m-restricted CTLs in CML patients were significantly higher than in healthy donors (P<0.01), but Flu-restricted CTLs had no variation in two groups, which indicated both peptides could induce cellular immune response. These epitopes can be used to develop vaccine-based active immunotherapy and CTL-based adoptive immune therapy. There was no significant difference between p210^bcr-abl642- specific CTLs and p210^bcr-abl926m-specific CTLs in CML patients. Percentages of peptide-positive CTLs were also compared in different clinical phases, the result displayed that p210^bcr-abl642 specific CTLs sharply decreased in blast-crisis phase compared with those in chronic phase (P=0.03). This phenomenon suggested that p210^bcr-abl642 specific CTL number may be useful in the diagnosis of CML with different stages.Part III: Generation of soluble HLA-A*0201-IgGFc fusion proteinThe total messager RNA was extracted from peripheral blood mononuclear cells with Trizol reagent. After reverse-transcription, cDNA of IgG Fc fragment was amplified with IgGFc specific primers. IgGFc gene fragment was inserted into pFastHTa vector, called pFHT-IgGFc. Next the gene fragment of soluble HLA-A*0201was cloned into pFHT-IgGFc, being located at the upstream of IgGFc. So the recombinant vector, pFHT-sHLA-A*0201-IgGFc, was expected to express a fusion protein, called sHLA-A*0201-IgGFc.The pFHT-HLA-A*0201-IgGFc plasmid was transformed into DH10Bac E.coli. The fusion gene fragment HLA-A*0201-IgGFc was transposed into bacmid with the screenment of blue or white colony. Next the recombinant bacmid DNA was transfected into Sf9 cells with liposome. Seventy-two hours later, the supernants of cell-culture that contained baculovirus were collected and then infected other Sf9 cells to express the fusion protein. The lysates of infected insect cells were identified by SDS-PAGE, and western-blot, and ELISA method, sequentially. Results showed that molecular weight of the recombinant fusion protein was 65kD, which is consistent with the expected size. In addition, the fusion protein could interact with the antibody (W6/32) which is against the classical class I HLA molecules, and bind to the antibody which is against the human IgG Fc fragment.In summary, we identified two epitopes from p210^bcr-abl (one in fusion region and another outside fusion region) according to three algorithms and the MHC stabilization assay on live T2 cells. Then these peptide-associated soluble HLA-A*0201 tetramers were generated to detect frequencies of peptides-specific CTL cells in CML patients. It was shown that there existed two peptide-specific CTLs in CML patients and p210^bcr-abl642-specific CTLs sharply decreased in blast-crisis phase. Finally, HLA-A*0201-IgGFc fusion protein was generated by baculovirus-insect cell expression system. This study not only laid foundations for further research on phenotype and function of the epitope-specific CTLs, but also opened the way to develop new strategies for immune therapies.