Yubing Zhou

Mutations increased overexpression of Notch

BackgroundThe Notch signaling pathway is crucial in T-cell development, Notch 1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To investigate the feature of Notch 1 mutation and its corresponding expression level in Chinese patients with T-ALL, detection of mutation and the expression level of Notch 1 gene was preformed using RT-PCR, sequencing and real-time PCR respectively.ResultsTwo Notch 1 point mutations (V1578E and L1593P) located on HD-N domain were identified in three cases out of 13 T-ALL patients. The mutation on 4733 position (V1578E) found in two cases was a novel mutation. The overexpression of Notch 1 was detected in all samples with T-ALL, moreover, significantly higher expression of Notch 1 was detected in the T-ALL with Notch 1 mutation group compared with T-ALL with WT Notch 1 group (p = 0.0192).ConclusionsHigher expression of Notch 1 was associated with Notch 1 mutation, more novel mutation of this gene might be identified in different populations and its contribution to the molecular pathogenesis of T-ALL is needed further research.

Clonal expanded TCR Vbeta T cells in patients with APL.

T-cell receptor Vss gene repertoire and clonality have been studied in patients with leukemia and solid tumors, by assaying the CDR3 size of TCR genes, using RT-PCR and genescan analysis. Few studies have studied leukemia-associated oligoclonal expanded T-cells in leukemia, therefore, the aim of this study was to investigate the distribution and clonal expansion of T-cell receptor, Vss subfamily T-cells in patients with acute promyelocytic leukemia (APL) with t(15;17). The CDR3 of TCR Vbeta24 subfamily genes were analyzed in peripheral blood mononuclear cells from 17 cases with PML-RARalpha+ APL using RT-PCR and genescan technique. Ten normal individuals served as controls. The results showed that the number of expressed Vss subfamilies (from 2 to 21 subfamilies) varied in different patients with APL. The most frequently expressed Vss subfamilies were Vbeta2 (64.7%), Vbeta15 (58.8%), Vbeta3 and Vbeta5 (47.1%), with a lower expression rate found in Vbeta11 and Vbeta20 (11.7%). Clonally expanded T-cells in the Vss subfamilies could be identified in patients with APL in all but two of the cases studied, predominantly in Vbeta10, Vbeta23, Vbeta3 and Vbeta21. In conclusion, skewed distribution and clonal expansion of TCR Vss subfamily T-cells could be found in patients with APL. The clonal expansion of T-cells were considered to be a specific anti-leukemic immune response by host T-cells activated by the leukemia-associated-antigen.

Mutations increased overexpression of Notch 1 in T-cell acute lymphoblastic leukemia

BackgroundThe Notch signaling pathway is crucial in T-cell development, Notch 1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To investigate the feature of Notch 1 mutation and its corresponding expression level in Chinese patients with T-ALL, detection of mutation and the expression level of Notch 1 gene was preformed using RT-PCR, sequencing and real-time PCR respectively.ResultsTwo Notch 1 point mutations (V1578E and L1593P) located on HD-N domain were identified in three cases out of 13 T-ALL patients. The mutation on 4733 position (V1578E) found in two cases was a novel mutation. The overexpression of Notch 1 was detected in all samples with T-ALL, moreover, significantly higher expression of Notch 1 was detected in the T-ALL with Notch 1 mutation group compared with T-ALL with WT Notch 1 group (p = 0.0192).ConclusionsHigher expression of Notch 1 was associated with Notch 1 mutation, more novel mutation of this gene might be identified in different populations and its contribution to the molecular pathogenesis of T-ALL is needed further research.

Generation of diffuse large B cell lymphoma-associated antigen-specific Vα6/Vβ13+T cells by TCR gene transfer

BackgroundOur previous study had amplified antigen-specific full-length TCR α and β genes of clonally expanded T cells in the peripheral blood (PB) of patients with diffuse large B-cell lymphoma (DLBCL). The transfer of T cell receptor (TCR) genes endows T cells with new antigen specificity. Therefore, the aim of this study is to generate diffuse large B cell lymphoma (DLBCL)-specific T cells by T cell receptor (TCR) gene transfer.Materials and methodsTwo different eukaryotic expression plasmids harboring TCR Vα6 and TCR Vβ13 genes specific for DLBCL-associated antigens were constructed and subsequently transferred into human T cells using Nucleofector™ technique. The expression of targeted genes in TCR gene-modified cells was detected by real-time PCR, and western blot using TCR Vβ antibody. The specific cytotoxicity of TCR gene-transferred T cells in vitro was estimated using a lactate dehydrogenase (LDH) release assay.ResultsTwo different eukaryotic expression plasmids harboring TCR Vα6 and TCR Vβ13 genes specific for DLBCL-associated antigens were constructed and subsequently transferred into T cells from healthy donors. Specific anti-DLBCL cytotoxic T lymphocytes (CTL) could be induced by transduction of specific TCR gene to modify healthy T cells. The transgene cassette of TCR Vβ13-IRES-TCR Vα6 was superior to the other in the function of TCR-redirected T cells.ConclusionsSpecific anti-DLBCL cytotoxic T lymphocyte (CTL) could be inducted by transduction of specific TCR gene to modify healthy T cells.

Enhancement of specific cellular immune response induced by DNA vaccines encoding PML-RARα and hIL-2 genes

Abstract A DNA vaccine encoding PML-RAR α fusion gene is thought to be a promising approach for acute promyelocytic leukemia patients to enhance immune responses after attaining complete remission. In this study, we sought to enhance cellular immunity by coexpressing human interleukin (hIL)-2 genes. Successfully constructed plasmids PML-RAR α-hIL-2-pIRES, PML-RAR α-pIRES and hIL-2-pIRES were delivered intramuscularly in BALB/C mice at 14-day intervals for three cycles. The cellular immune responses with respect to the specific cytotoxicity of spleen cells; interferon-γ secretion in sera, and the T-cell receptor rearrangement excision circles of thymocyte were significantly increased from PML-RARα-hIL-2-pIRES immunized mice. Our results indicate that a DNA vaccine with PML fusion gene segment and hIL-2 together might elicit increased cellular immune responses in mice.