Identification of HLA-A2 restricted cytotoxic T lymphocyte epitopes from tumor antigen PIWIL2

AIM: To identify the human leucocyte antigen A2 (HLA-A2) restricted cytotoxic T lymphocyte (CTL) epitopes from tumor antigen PIWIL2. METHODS: RT-PCR and Western blot was used to determine the expression of PIWIL2 in cancer cell lines MCF-7, SW480 and HT-29. HLA-A2 epitopes from PIWIL2 protein were predicted by the software of BIMAS, RankPep, NetMHC, NetCTL1.2 and IEDB. The peptides were synthesized by standard solid-phase methods. The binding affinity of the peptides to HLA-A2 molecules was evaluated by T2 cells binding assay. ELISPOT assay was used to investigate the levels of IFN-γ. The cytotoxicity assay in vitro was also used to determine the ability of inducing T cell response by the peptides. RESULTS: The expression of PIWIL2 was observed in MCF-7, SW480 and HT-29. The candidate peptide P485, P493 and P965 showed moderate affinity toward HLA-A2 molecule. ELISPOT assay showed P485 and P965 induced CTLs of IFN-γ release form CTLs. The CTLs induced by P485 and P965 lysed the MCF-7 cells. CONCLUSION: The peptides P485 and P965 are excellent HLA-A2 restricted cytotoxic T lymphocyte epitopes from the tumor antigen PIWIL2, which could serve as new candidates towards antitumor peptide vaccines.     

Design and immune activity detection of long peptide vaccines targeting lung cancer antigen COX-2

AIM:To design long peptides based on cytotoxic T-lymphocytes (CTL) epitope prediction for lung cancer antigen cyclo-oxygenase-2 (COX-2) and to study the immune activity of the long peptides. METHODS:HLA-A2 epitopes from COX-2 protein were predicted by NetCTL 1.2, SYFPEITHI and IEDB. The CTL epitope-concentrated area was analyzed, and the appropriate length of long peptides were designed. In vitro activity experiments were used to verify the immune activity of the long peptides. ELISPOT assay and intracellular cytokine staining assay were used to investigate the ability of the peptide to induce specific restricted CTLs and release of interferon-γ (IFN-γ). The ability of the peptides to induce T-cell response was investigated by lactate dehydrogenase (LDH) and CFSE cytotoxicity assay in vitro. RESULTS:ELISPOT and intracellular cytokine staining assay showed P315-338 and P375-401 were able to induce specific CTLs and higher levels of IFN-γ release. The results of LDH and CFSE cytotoxicity assays showed the CTLs induced by P315-338 and P375-401 lysed the target cells. CONCLUSION:Two long peptides pointing to lung cancer antigen COX-2 are successfully identified, which could be used as immunotherapy vaccine in future.     

Identification of HLA-A3 restricted cytotoxic T-lymphocyte epitopes from cancer-testis antigen MAGEC2

AIM:To predict and identify an HLA-A3 supertype-restricted cytotoxic T-lymphocyte (CTL) epitope derived from MAGEC2, which is utility in epitope design for the development of HLA-based vaccines and immunotherapeutics. METHODS:HLA-A3 epitopes from MAGEC2 protein were predicted by BIMAS, SYFPEITHI and IEDB. The binding affinity of the peptides to HLA-A*03 molecule was evaluated by T2A3 cell binding assay. ELISPOT assay was used to investigate the ability of the peptides inducing specific restricted CTLs to release interferon-γ (IFN-γ). The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro. RESULTS:The candidate peptides P147, P167, P196, P229 and P251 showed moderate affinity toward HLA-A3 molecule. ELISPOT assay showed that P167, P196 and P251 were able to induce specific CTLs and higher levels of IFN-γ were released. The CTLs induced by P196 and P251 were able to lyse target cells. CONCLUSION:The peptides P196 and P251 have higher binding affinity with HLA-A3 and retain immunogenicity. They are excellent HLA-A3-restricted CTL epitopes from tumor antigen MAGEC2, which could serve as new candidates towards antitumor peptide vaccines.     

Identification and molecular modification of cytotoxic T-lymphocyte epitopes from osteosarcoma high-expressing antigen PBF

AIM: To observe whether modified epitopes from osteosarcoma high-expressing antigen papillomavirus-binding factor (PBF) have HLA-A2 restricted antitumor ability, and to develop peptide-based immunotherapy for osteosarcoma. METHODS: RT-PCR and Western blot were used to determine the expression of PBF in the osteosarcoma cell lines U2OS and Saos-2. HLA-A2 epitopes from PBF protein were predicted by NetCTL 1.2, SYFPEITHI and IEDB. The modified peptides from PBF containing HLA-A2 binding anchor motifs were designed by replacing the anchor residues. The peptides were synthesized by standard solid-phase methods, and the binding affinity of the peptides to HLA-A*0201 was evaluated by T2A2 cell binding assay. ELISPOT assay was used to investigate the seretion of interferon-γ (IFN-γ) from the peptide-induced specific cytotoxic T-lymphocytes (CTLs). The ability of inducing T-cell response was analyzed by lactate dehydrogenase (LDH) release assay and carboxyfluorescein succinimidyl ester (CFSE) cytotoxicity assay in vitro. RESULTS: The expression of PBF was observed in the U2OS and Saos-2 cells. The candidate peptides P75-1Y2L, P412-1Y, P416-1Y2L9V, P107-1Y and P435-1Y2L showed moderate affinity toward HLA-A2 molecule. The modified peptides showed significantly higher affinity with HLA-A2 than the native peptide. ELISPOT assay showed that P412, P412-1Y, P416, P416-1Y2L9V and P435-1Y2L induced specific CTLs to secrete IFN-γ, and P412-1Y and P416-1Y2L9V induced more secretion of IFN-γ than the native peptide. The CTLs induced by P412, P412-1Y, P416 and P416-1Y2L9V lysed U2OS cells. P412-1Y and P416-1Y2L9V peptide-specific CTLs showed higher cytotoxicity against U2OS cells than the native peptide-specific CTLs. CONCLUSION: Compared with the native peptide, modified epitopes P412-1Y and P416-1Y2L9V have higher binding affinity with HLA-A*0201 and retain immunogenecity. In addition, the anti-tumor immunity effects of modified epitopes P412-1Y and P416-1Y2L9V are stronger than the native peptide. The peptides P412-1Y and P416-1Y2L9V is excellent HLA-A*0201 restricted CTL epitopes from tumor antigen PBF, which could serve as new candidates towards antitumor peptide vaccines.     

Identification of cytotoxic T-lymphocyte epitopes from hepatocellular carcinoma antigen MAGEC2

AIM: To observe whether modified epitopes from hepatocellular carcinoma antigen MAGEC2 have HLA-A2-restricted antitumor ability. METHODS: HLA-A2 epitopes from MAGEC2 protein were predicted by NetCTL 1.2, SYFPEITHI and IEDB. The change of binding anchor motifs by replacing anchor residues created the modified peptides from MAGEC2. The binding affinity of the peptides to HLA-A*0201 molecule was evaluated by T2 cells binding assay. ELISPOT assay and intracellular cytokine staining were used to investigate the ability of the peptides inducing specific restricted cytotoxic T-lymphocytes (CTLs) to release interferon-γ (IFN-γ). The ability of the peptides to induce T-cell response was investigated by cytotoxicity assay in vitro. RESULTS: The candidate peptides P248, P248-1Y, P356, P356-1Y, P356-2L and P356-1Y2L showed moderate affinity toward HLA-A2 molecule. T2 binding assay showed that P248-1Y and P356-1Y2L showed significantly higher affinity for HLA-A2 than the native peptides. ELISPOT assay and intracellular cytokine staining showed P248, P248-1Y, P356 and P356-1Y2L were able to induce specific CTLs to release IFN-γ. ELISPOT assay showed that significantly higher levels of IFN-γ release were induced by P248-1Y and P356-1Y2L than the native peptides. The CTLs induced by P248, P248-1Y, P356 and P356-1Y2L lysed HepG2 cells, and P248-1Y and P356-1Y2L peptide-specific CTLs showed higher cytotoxicity against HepG2 cells than the native peptide-specific CTLs (P<0.05). CONCLUSION: Compared with the native peptides, modified epitopes P248-1Y and P356-1Y2L have higher binding affinity with HLA-A2 and retain immunogenecity. In addition, the antitumor immunity effects of modified epitope P248-1Y and P356-1Y2L are stronger than the native peptides. The peptides P248-1Y and P356-1Y2L are excellent HLA-A2-restricted CTL epitopes from tumor antigen MAGEC2, which could serve as new candidates towards antitumor peptide vaccines.     

Prediction and identification of B-cell epitopes of TIMP-1

AIM: To predict and identify the B-cell epitopes of human tissue inhibitor of metalloproteinase-1 (TIMP-1). METHODS: The prediction of B-cell epitopes of TIMP-1 was conducted by DNAStar and BcePred softwares, and 8-branch form of multiple antigen peptide (MAP) was synthesized, which were subsequently used for immunizing rabbits mixed with an universal T-helper epitope human IL-1β peptide (VQGEESNDK, amino acid 163~171). The immunogenicity of the synthesized peptide was evaluated by monitoring the antiserum titer and analysis of binding affinity with antibody. The specificity of produced antiserum was detected by Western blotting. RESULTS: Amino acids 27~41 (MAP1), 57~71 (MAP2),95~109 (MAP3) and 193~207 (MAP4) of TIMP-1 were predicted as the most potential epitopes and synthesized as immunogen. MAP2, MAP3 and MAP4 induced antibodies with high titer by the dynamic monitoring, which was also identified as specific antibody against TIMP-1 by Western blotting. Affinity analysis showed MAP4 had the highest binding activity with market antibody. CONCLUSION: The No. 57~71 and 193~207 peptides of TIMP-1 protein are proved to be the preponderant B-cell epitopes and the 193~207 epitope is believed to have the strongest immunogenicity, which may assist the development of therapeutic approach against high expression of TIMP-1.     

国际葡萄基因组学学术研讨会在美国圣路易斯市召开

国际葡萄基因组学学术研讨会(INternational Grape Genomics Symposium)于2005年7月12~14日在美国密苏里州圣路易斯市召开。本次会议由西南密苏里州立大学(SMSU)主办,国际葡萄基因组计划(International Grape Ge-nome Program)和美国葡萄栽培与酿酒学会东部分会(ASEV-ES)协办。     

Induction of tumor-specific cytotoxic T lymphocytes in vitro by dendritic cells pulsed with different glioma antigens

AIM: The goal of this study was to compare different methods for tumor antigen preparation, to observe the induction of tumor-specific cytotoxic T lymphocytes in rats by dendritic cells (DCs) pulsed with different tumor antigens. METHODS: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thaw, boiling or total protein extracted from ultrasonic crushed tumor cell. Subsequently primed DCs were cocultured with T lymphocytes isolated from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-γ release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test. RESULTS: DCs pulsed with boiled tumor cell in vitro induced an enhanced ability of T-cell proliferation and cytotoxic T lymphocyte activity.CONCLUSION: Our results demonstrated that DCs primed with boiled tumor cell may represent a method for inducing immune responses against the entire repertoire of tumor antigens of malignancies.     

Advances in research of phage peptide library

Phage peptide libraries are collections of the specific length of short peptides, and they are based on phages as their carriers. They include mimotope libraries and pept ide antibody libraries. Phagedisplayed pept ide libraries have been used to isolate specific ligands for numerous protein targets, and they have been proven useful in defining antigen momitopes, rapid determination of binding energetics at protein-protein interfaces, designing of vaccine and tumor research aspects. This review summarized the research progression of phage peptide library.     

茉莉酸甲酯诱导葡萄悬浮细胞防卫反应机制的研究

为明确茉莉酸甲酯(methyl jasmonate,Me JA)诱导葡萄果实抗病反应的机制,以‘巨峰’葡萄悬浮细胞为试材,将其继代培养一次后分别在含10μmol·L-1 Me JA的B5培养基和含100 nmol·L-1隐地蛋白的B5培养基中(模拟病原菌接种)振荡培养15 d,每3 d测定1次细胞质量及抗病相关指标。结果显示:单一Me JA处理未激活葡萄悬浮细胞内的防卫反应;而单一隐地蛋白处理则可立即显著诱导葡萄悬浮细胞内源H2O2迸发,提升vv NPR1.1、PR1和PR2表达水平和植保素含量;经Me JA处理的葡萄悬浮细胞在添加隐地蛋白后,其细胞内出现较单一隐地蛋白处理更为显著的H2O2迸发、PR基因表达和植保素合成现象,说明经Me JA处理的悬浮细胞只在病原激发子胁迫时才表达出较强的系统抗性。因此,Me JA诱导的葡萄悬浮细胞抗病反应可归因于Priming(植物敏化过程或防御准备过程)机制。此外,经Me JA诱导的Priming反应对葡萄悬浮细胞生长无显著影响,暗示其未抑制葡萄细胞生长和胞内物质积累。     

“2012年园艺植物染色体倍性操作与遗传改良学术研讨会”预备通知

自2008年11月在中国农业科学院郑州果树研究所成功召开园艺植物染色体倍性操作与遗传改良研讨会以来,我国科研人员在该领域的研究取得了可喜的成绩。为进一步总结近几年来在该领域的研究进展,促进园艺植物倍性育种研究,同时为该领域的专家、学者和同仁们提供良好的交流平台。中国园艺学会定于2012年4月中旬在重庆召开园艺植物染色体倍性操作与遗传改良学术研讨会。欢迎从事该研究领域及相关研究工作的人员参加。     

Establishment and assessment of a method for acid elution of platelet HLA class I antigens

AIM: To establish an effective method for acid elution of platelet HLA class I antigens, and to evaluate the optimal condition and the feasibility of clinical application of the acid-elution technique. METHODS: Platelets were treated with citric acid buffer at different pH levels (pH=2, 3, 5, 7). Expression of HLA class I antigens and P-selectin (CD62P) on the platelet surface was analyzed by multicolor flow cytometry. The proportion of early apoptotic platelets was detected by Annexin V staining. The maximum platelet aggregation rate was determined by electrical impedance aggregometry.RESULTS: With the decrease in the pH levels of the citric acid buffer (from pH=7 to pH=2), the expression of HLA class I antigens on the platelet surface was remarkably decreased. However, the rates of platelets activation (CD62P expression) and early apoptosis (Annexin V expression) were both significantly increased. Compared with PBS, treatment of the platelets with citric acid buffer at pH 3.0 remarkably reduced the expression of platelet HLA-class I antigens (P<0.05). Although the rates of the platelet activation and apoptosis were also significantly increased (P<0.05), the aggregation of platelet was not remarkably reduced (P>0.05).CONCLUSION: Acid elution of platelet HLA-class I antigens with citric acid buffer at pH 3.0 at 0 ℃ can be use as an attempt to produce HLA-eluted platelets. This technique of acid-elution needs further improvement and standardization before clinical use.     

Cloning,expression, biological characteristics and tissue localization of a Clonorchis sinensismembrane antigen/excretory-secretory antigen,acid phosphatase

AIM:To clone, express and determine the biological characteristics and tissue localization of Clonorchis sinensis(C. sinensis, Cs) acid phosphatase (AP), and to identify CsAP as C. sinensismembrane antigen/excretory-secretory antigen. METHODS:The methods of bioinformatics, molecular biology, immunohistochemistry and gelatin zymography were used to analyze CsAP. RESULTS:From cDNA library, a full-length 1 410 bp cDNA clone of Cs encoding a novel AP was selected, recombinated, expressed and purified in Escherichia coli. The molecular weight of the CsAP recombinant was 55 kD. CsAP was a membrane antigen as well as an excretory-secretory antigen. The fluorescence of CsAP was located in metacercaria and intestinal cecum and tegument of adult, but not detected in redia or cercaria. The cross reactions between Clonorchis sinensis and Schistosoma japonicum in the recognition of their infective sera by CsAP was observed. The recognition of CsAP with C. sinensiscrude antigens was not different among the mild, midrange or grave C. sinensisinfection. After immuned by CsAP recombinant, the total IgG antibody titer of the rats went up to the peak value of over 1∶25 600 in the 3rd week. CsAP had the ability to degrade collagen. CONCLUSION: CsAP is highly expressed in Escherichia coli, showing its immunogenicity, but not a good diagnostic marker, and might be an excretory-secretory protein as well as a membrane protein.     

Screening of tissue factor targeting peptides alternately from phage display peptide library

AIM: To screen tissue factor (TF) targeting peptides by establishment of a new and effective phage display method to acquire the peptides specifically bound to the transmembrane receptor. METHODS: Five rounds of panning were alternately conducted by targeting TF and HT-29 cell line which showed the detectable TF expression (screen for targeting receptor and cell alternately with phage display, STRCA). The 30 phage clones were assessed by enzyme-linked immunosorbent assay (ELISA). DNA sequencing was performed for the phage clones. The affinity of synthetic peptides was verified with competitive inhibition ELISA. The repeated experiment was conducted to verify the reliability of the results. RESULTS: The phages were effectively enriched after 5 rounds of panning with the improvement of the recovery rate from (2.25×10^-4)% to (1.32×10^-2)%. In 30 individual phages, ELISA positive rate was 76.7%, and the repetition of A, B, C and D peptides showed 23.3% (7/30), 23.3% (7/30), 26.7% (8/30) and 10.0% (3/30),respectively. E peptide constructively consisted of A and B. The five synthetic peptides were verified by ELISA, and the IC₅₀ of each peptide showed 3.25 nmol/L, 6.72 mol/L, 3.24×10³ mol/L, 2.08×10² mol/L and 45.77 mol/L,respectively. The positive phages were selected again in the second experiment to compare the results of the first experiment and the repeatability was 33.3%. CONCLUSION: STRCA can select TF targeting peptides with high affinity, which has the potential to become a therapeutic screening of transmembrane receptor-binding peptides.     

Selection of targeted glioblastoma tumor cell-binding and internalizing peptides through phage display vector

AIM: To isolate peptides targeted binding and internalizing into glioblastoma cell line SWO-38. METHODS: Tumor cells were screened five rounds of whole cell screen through the Ph.D.-12 phage display library. The monoclone specific binding efficiency to the tumor cell was analyzed, and the DNA of phages were extracted, sequenced and translated to the sequences of amino acid. RESULTS: In the phage library after five rounds of screen , 10 of 13 monoclones had highly selective binding to SWO-38 cells. We found two repeated peptide sequences. CONCLUSION: Whole cell screening against tumor cells through random phage peptide library can obtain phage peptides with highly specific binding and internalizing ability. The peptides could be used as a therapy vector for tumor targeted delivery.     

In vivo immunogenic characteristics of cytotoxic T lymphocyte epitopes from pancreatic tumor antigen MUC4

AIM: Peptide vaccines have been conceived as promising therapies for tumor-inflicted patients due to their easy production and capability of inducing specific immune response required for defending the tumor. During our previous research, 4 HLA-A2-restricted peptides had been identified as immunogenic in vivo. In this study, we aimed to testify the in vivo immunogenicity of the 4 peptides. METHODS: BALB/c mice were vaccinated with HLA-A2 restricted peptides emulsified in incomplete Freund's adjuvant (IFA) subcutaneously in combination with the epitope at the adjacent location. After the 3rd peptide vaccination for 10 d, the peptide-specific immune response was evaluated by ELISPOT and ELISA. The ability to induce T cell response was investigated by using cytotoxicity assay in vivo and the presence of peptide-specific CD8⁺ T cells capable of recognizing the MHC-peptide was detected by flow cytometry. RESULTS: Among the 4 candidate HLA-A2 restricted peptides, the immune response elicited by P2004-1Y9V was superior to that of the other 3 peptides. The CTLs induced by P2004 and P2004-1Y9V lysed CAPAN-2 cells. P2004-1Y9V peptide-specific CTLs showed higher cytotoxicity against pancreatic tumor cell lines of CAPAN-2 than the native peptide-specific CTLs. Intracellular cytokine staining assay indicated the presence of P2004-1Y9V specific CD8⁺T cells in the P2004-1Y9V vaccinated mice. CONCLUSION: P2004-1Y9V is the most immunogenic peptide in vivo, and can be explored as potential tumor peptide vaccine in the future clinical study.     

Screening of lung cancer specific peptide ZS-9 from a phage display peptide library and NCI-H1299 cells

〓AIM: To obtain the polypeptides specifically bound to NCI-H1299 cell line from peptide libraries and to identify these polypeptides affinity and specific to lung cancer. METHODS: The lung cancer NCI-H1299 cell line was used as the antigen and MRC-5 was used as control for subtraction biopanning from a phage display peptide library. The positive and specific binding clones were identified by cell enzyme-linked immunosorbent assay (ELISA) and immunochemistry staining. Those DNA sequences of identified clones were sequenced, and the amino acid sequence was deduced and analyzed with bioinformatics. RESULTS: After 3 rounds of panning, 9 phage clones were identified by ELISA, one of them specially bound to the NCI-H1299 and A549. The DNA sequence result of ZS-9 was CAT AAT AAG CAT CTT CCG TCT ACG CAG CCT CTT GCG. Hence, the polypeptide sequence was HNKHLPSTQPLA deduced from its DNA sequence. The amino acid sequence was analyzed in NCBI and Swiss-Prot, the results showed that it has no similarity with the known proteins sequence in the database. All these results showed that we have discovered a novel lung cancer surface associated antigen ligand. CONCLUSION: A peptide which is specific binding to lung cancer cell line NCI-H1299 and A549 has been selected from phage display peptide libraries. Therefore, it provides a potential tool for early diagnosis of lung cancer or targeted drug delivery in chemotherapy.     

Application of molecular recognition theory in thyrotropin and its receptor system

AIM: The selective recognition of the sense peptides which are located in special regions of thyrotropin receptor (TSHR) by their corresponding antisense peptides has been investigated. Three pairs of sense and antisense peptides were named TR1 (aa37-45) and RT1 (aa45-37), TR2 (aa353-366) and RT2 (aa366-353), TR3 (aa648-655) and RT3 (aa655-648). METHODS: To prepare three affinity chromatography columns, antisense peptides were immobilized, called RT1-sepharose 4B, RT2-sepharose 4B and RT3-sepharose 4B, respectively and investigate the retardative behavior for each of native peptide TR1, TR2 or TR3 on above columns with stepwise elution. RESULTS: Each of the three immobilized antisense peptides recognized and retarded its corresponding sense peptide-TR1, TR2 or TR3 instead of those non-complementary peptides. Immobilized RT1 recognized free TSHR protein molecule as well. In additional, bovine thyrotropin was recognized by immobilized TR1. CONCLUSION: The results indicate that molecular recognition theory exsits in thyrotropin receptor system. It may be useful to isolate biological molecules and to locate epitopes of TSH on TSHR molecule. Otherwise, antisense peptide may be used for treatment of experimental autoimmunolized thyroid disease (AITD) in the rat.     

Relationship between helper T lymphocytes immune deviation and class Ⅱ MHC antigen expression in acute rejection of transplanted heart

AIM: To observe the relationship between the immune deviation of Th1 and Th2 cell clones and class Ⅱ major histocompatibility complex (MHC) antigen expression in different stages of acute rejection in transplanted hearts. METHODS: Heart transplantation were performed in rats.Isografts and non-transplanted animals were used as control group. Donor class II MHC antigen expression were detected with monoclonal antibodies and immunostaining technique and the amount of type Ⅰ and Ⅱ cytokines mRNA expression were detected by semiquantitative RT-PCR in cardiac allografts. RESULTS: Myocardial IL-2 mRNA and donor class Ⅱ MHC antigen expression were significantly in-creased, accompanied with development of acute rejection (P<0.01). However, IL-4 mRNA decreased significantly (P<0.01). The immune deviation between type Ⅰ and Ⅱ cytokines mRNA expression occurred in the rejecting stage at which the cardiac allograft tissue infiltrated by multiple foci of lymphocytes and class Ⅱ MHC antigen became highly expressed. CONCLUSION: There was correlationship between Th cells immune deviation and donor class Ⅱ MHC antigen expression in the process of acute rejection in cardiac allografts. Immune deviation of T cells may facilitated the expression of donor MHC class Ⅱ antigen on transplanted heart.     

The inducement,restricted usage and clonal expansion of TCR Vβ subfamily T cells related to chronic myelogenous leukemia

AIM: To investigate the anti-leukemia effect, the restricted usage and clonal expansion of TCR Vβ subfamily T cells from donor peripheral blood induced by chronic myelogenous leukemia(CML) cells, K562 cells and bcr-abl peptide, respectively. METHODS: T cells in donor's peripheral blood were stimulated with CML cells, K562 cells and bcr3-abl2 peptide and amplified by MLTC, to induce the CML specific cytotoxic T lymphocytes. The induced T cells were further analyzed for the restricted usage and clonal expansion of TCR Vβ subfamilies by using RT-PCR and genescan analysis, and the detection of specific cytotoxicity in CML by LDH release assay. RESULTS: 10-13 Vβ subfamilies were expressed in T cells from donor peripheral blood which were induced with CML cells, K562 cells and bcr-abl peptide in 1-2 weeks by MLTC. Oligoclonal T cell in Vβ16, Vβ21 and oligoclonal tendency T cells in Vβ5, Vβ13 subfamilies were identified in induced T cells, which have the ability of specific cytoxicity to CML cells and K562 cells. CONCLUSION: The anti-CML cytotocity T cells were induced by CML cells, K562 cells and bcr-abl peptide. These induced T cells with specific cytoxicity effect may come from the clonal expansion TCR Vβ subfamily T cells.