| Chronic myeloid leukemia (CML) is a malignant proliferative disorder of pluripotential hemopoietic stem cell by origin and characterized by the unique Philadelphia (Ph) chromosome that results in the bcr/abl fusion oncogene and the expression of a chimeric protein product BCR/ABL. The unique amino acid sequence in junctional region of the BCR/ABL represents a potential CML-specific antigen. It has been confirmed recently that peptides crossing the b3a2 fusion breakpoint in p210 can be presented on the cell surface, within the cleft of human leukocyte antigen (HLA) molecules I and II and be recognized by cytotoxic T lymphocytes (CTL) and helper T cell (TH) respectively. Although an advantageous foundation has been provided in immunotherapy with CML, effective immune response can hardly be stimulated in some CML patients because of immunological dysfunction, including deficiency in co-stimulation molecule B7 and HLA molecule. Therefore, methods of activating CTL in vitro are being explored. Although the methods of gene transfection or peptide loading APC etc have been investigated successfully in vitro, their applications are limited by the defects themselves.-6-Previous studies have demonstrated that an arginine-rich peptide TAT48-60 (GRKKRRQRRRPPQ), named protein transduction domain (PTD), derived from the human immunodeficiency virus (HIV)-l TAT protein has the ability to translocate through the cell membranes. Although the mechanism of protein transduction is not clear hitherto, PTD can rapidly and efficiently enter into cells and are independent of receptor- or endosome-mediated endocytosisIn this study, we describe the use of PTD-mediated delivery of exogenous soluble BCR/ABL into endogenous and exogenous antigen presentation pathways of APCs to activate antigen-specific CTL and TH in vitro.In the first part of our experiments, we incubated HL-60, C2C12 and SMMC-7721 cells with PTD-BCR/ABL fusion protein and then detected intracellular PTD-BCR/ABL staining by immunohistochemistry, examined mean fluorescent intensity of intracellular PTD-BCR/ABL and positive ratio of cells with various incubation times by flow cytometry (FCM). In addition, the purified PTD-BCR/ABL fusion protein as well as Evans blue were injected into mice through caudal vein and the cryostat sections of brain, liver, spleen, heart and kidney tissues were prepared 6h later and the distribution of PTD-BCR/ABL fusion protein in various tissues was observed by immunohistochemistry staining.In the second part of our experiments, The PBMCs from CML patients were stimulated in vitro with purified PTD-BCR/ABL antigen and then the expressions of the earlier activation antigen CD69 on CD8+ and CD4+ T cells after stimulation were detected by flow cytometry (FCM). BALB/C mice were vaccined with PTD-BCR/ABL and BCR/ABL antigen for 4 times and then the immune serum was collected at various time points and the value of optical densit (A ) of antibody was assayed by ELISA.-7-The results were as follows:(1) PTD-BCR/ABL was detected in cytoplasm and nucleus, especially obviously in nucleus, by immunohistochemistry staining after incubating HL-60, C2C12 and SMMC-7721 cells with PTD-BCR/ABL. However, no positive staining was detected in BCR/ABL group as control(2) The positive ratio of HL-60 cells with intracellular PTD-BCR/ABL was keeping higher during the 72h of transdution. It reached to 77% at 1.5h and 94.52% at 3h. The mean fluorescent intensity of intracellular PTD-BCR/ABL showed obvious changes following various times. During the initial stage of transdution, it kept increasing and reached to the utmost at 6h. Subsequently, it kept decreasing and was less of the half of the utmost at 72h.(3) PTD-BCR/ABL fusion protein was detected in brain, liver, spleen, heart and kidney tissues of mice after intravenous injection with it and it's distribution in various tissues was not specific. No Evans blue was detected in brain.(4) After stimulation with 100 mg/L of PTD-BCR/ABL antigen (final concentration... |
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