Analysis Of T-cell Receptor Vβ Gene Idiotype In ALL Patients And Develop DNA Vaccine

Objective: To investigate the situation of TCR Vβ gene repertoire and clonal expansion in peripheral blood T cells from patients with acute lymphoblastic leukemia (ALL ) and T cell strains, and analysis the expressive characters of TCR V β gene idiotype. To develop the anti-lymphoblastic leukemia TCR idiotypic DNA vaccine.Methods: The complementarity determining region 3 (CDR3) of TCR Vp 24 subfamily genes in peripheral blood mononuclear cell from 6 T cell strains as well as 6 cases with untreated T-ALL and 13 cases with untreated B-ALL were amplified using RT-PCR. The PCR products were further labelled by fluorescein and then analyzed by genescan technique for detection of the CDR3 size, to determine clonality of T cells. Some monoclonal PCR products from T cell strains were analyzed by sequencing to define the sequence of CDR3. The rearranged idiotypic CDR3 fragments coding TCR Vβ8 of the Jurkat cell strain and TCR Vβ2 of the Molt4 cell strain were amplified using RT-PCR, and then were cloned into pIRES vector respectively. The recombinant plasmids were analyzed by sequencing and the one containing the correct fragment was transfected into K562 cells. The condition of idiotypic protein expression was tested by Western-Blot.Results: 5~12 Vβ subfamilies were identified in 6 T-ALL cases and 2~18 Vβ subfamilies were identified in 13 B-ALL cases, respectively. Clonal expanded T cells, including oligoclonal, oligoclonal trend, bioclonal and monoclonal patterns, in one or more Vβ subfamilies were found in all cases. The display frequency of clonal expansive T cells in Vβ21 and Vβ23 subfamilies were higher than others in B-ALL cases. Only one monoclonal expanded Vβ subfamily T cell could be identified in the most T cell strains. Sequences from different T cell strains displayed different CDR3 length and sequences. The recombinant plasmids, pIRES-Jurkat Vβ8 and pIRES-Molt4 Vβ2①/②, containing idiotypic TCR Vβ8 frgment of Jurkat cell strain or idiotypic TCR Vβ2 frgments of the Molt4 cell strain were developed. But only pIRES-Molt4 Vβ2② was identified to containe the correct fracment by sequencing. A 15 KD protain which can bind with TCR Vβ2 antibody specialy were identified after pIRES-Molt4 Vβ2② was transfected into K562 cells.Conclusions: Restrictive changes of TCR Vp repertoire and clonal expanded T cells could be found in peripheral blood T cells from patients with T-ALL. However, the clonal expanded T cells' property (clonal expansion leukemic cells or leukemia antigen-specific expansion T cells) should be further characterized. It may play a certain role on detection of minimal residual disease and design of anti-leukemia idiotypic vaccine. The dominant utilization of TCR VjJ repertoire could be found in peripheral blood T cells from patients with B-ALL, meanwhile clonal expansive T cells were existed. It may be a feature of the host immune response for leukemia-associated antigen. The clonal expansive tendency of T cells in V021 and VP23 subfamilies was rather obvious, which may be correlated with certain malignant B-cell clone. The CDR3 sequences of monoclonal expansive T cell in T cell strains were different, which was the base of developing DNA vaccine. The recombinant plasmid pIRES-Moh4 Vp2d) was developed successfully, which can express special TCR VfJ2 protain in vitro.