Xianfeng Zha

Altered expression pattern of miR-29a, miR-29b and the target genes in myeloid leukemia

ObjectivesThe miR-29 family have been demonstrated acting as vital tumor suppressor in multiple cancers as well as regulators in the adaptive immune system. Little is known about their role in leukemogenesis. The purpose of this study is to analyze the expression pattern of miR-29a/29b and its target genes Mcl-1 (myeloid cell leukemia sequence 1) and B-cell lymphoma 2 (Bcl-2) in myeloid leukemia.MethodsQuantitative real-time PCR was used for detecting genes expression level in peripheral blood mononuclear cells (PBMCs) from 10 cases with newly diagnosed, untreated acute myeloid leukemia (AML) and 14 cases with newly diagnosed, untreated chronic myeloid leukemia (CML) in chronic phase, and 14 healthy individual (HI) served as controls. Correlation between the relative expression levels of different genes have been analyzed.ResultsSignificant lower expression of miR-29a/29b and higher expression level of two potential target genes Bcl-2 and Mcl-1 were found in PBMCs from AML and CML patients compared with HI group. In addtion, miR-29a expression in AML was significantly lower than that in CML. Moreover, negative correlation between miR-29a/29b and its target genes have been found. While, positive correlation between relative expression level of miR-29a and miR-29b or Bcl-2 and Mcl-1 were presented in the total 38 research objects.ConclusionDown-regulated miR-29a and miR-29b, and accompanying up-regulated Bcl-2 and Mcl-1 are the common feature in myeloid leukemias. These data further support the role for miR-29a/29b dysregulation in myeloid leukemogenesis and the therapeutic promise of regulating miR-29a/29b expression for myeloid leukemia in the future.

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.

Deficiency of CD3gamma, delta, epsilon, and zeta expression in T cells from AML patients

Abstract In order to elucidate the feature of T‐cell receptor (TCR) signal transduction in T‐cells from acute myeloid leukemia (AML), the expression levels of CD3gamma, delta, epsilon and zeta chain genes in CD3+ T cells were analyzed using real‐time PCR. CD3+ T cells sorted from peripheral blood of 10 AML patients and 10 healthy donors were used in the study. Significantly lower expression levels of all four CD3gamma, delta, epsilon, and zeta chain genes were found in the AML samples. The expression pattern of the four CD3 chains was epsilon>gamma>delta>zeta in CD3+ T cells from AML samples, which was different from the healthy control group. In conclusion, the results provide a global gene expression profile of CD3gamma, delta, epsilon, and zeta chains in AML patients. Deficiency of all four CD3 gene expression levels might represent the feature related to T‐cell immunodeficiency.

Differential gene expression profiles of PPP2R5C-siRNA-treated malignant T cells.

Recently, alterations in the expression pattern of PPP2R5C associated with malignant transformation have been characterized, and PPP2R5C overexpression was demonstrated in leukemias. To confirm the role of PPP2R5C in proliferation and its molecular mechanism, three PPP2R5C-siRNAs and a scrambled nonsilencing siRNA control were used to treat Molt-4 and Jurkat T cells. After nucleofection, PPP2R5C expression and biological consequences based on a highly efficient and specific PPP2R5C-siRNA were demonstrated by qRT-PCR, CCK-8 assay, Annexin V/PI, and flow cytometry. The global gene expression profile of PPP2R5C-siRNA-treated Jurkat T cells was established. A significant reduction in the PPP2R5C mRNA level was observed at 24 to 72 h in Molt-4 and Jurkat T cells with all of the PPP2R5C-siRNAs. The proliferation rate of Molt-4 and Jurkat T cells transfected with different PPP2R5C-siRNAs was significantly decreased at 72 h compared with the control (p<0.05). However, the transfected cells did not show a significant increase in Annexin V/PI-positive cells (apoptosis). The highly efficient PPP2R5C-siRNA2 was used to treat Jurkat T cells for gene expression profile analysis. In total, 439 genes were upregulated, and 524 genes were downregulated at least twofold in PPP2R5C-siRNA-treated Jurkat T cells. Changes in signaling pathway genes closely related to the TCR, Wnt, calcium, MAPK, and p53 signaling pathways were observed. In conclusion, the suppression of PPP2R5C by RNA interference could effectively inhibit the proliferation of leukemic T cells, the PPP2R5C-siRNA treatment altered gene expression profiles, and the differential expression of the glycogen synthase kinase 3 beta (GSK-3β), ataxia telangiectasia mutated (ATM), and Mdm2 p53 binding protein homolog (MDM2) genes may play an important role in the effects of PPP2R5C knockdown in Jurkat T cells.

Upregulated TCRζ Enhances Interleukin-2 Production in T-Cells from Patients with CML

T-cell immunodeficiency is a common feature in patients with chronic myeloid leukemia (CML), and deficiency in CD3 levels was detected in T cells from these patients, which may represent a characteristic that is related to a lower T cell activation. In this study, we explored the possibility that forced TCRζ gene expression may upreg-u-late T cell receptor (TCR) signaling activation and reverse interleukin-2 (IL-2) production in T cells from patients with CML. A recombinant eukaryotic vector expressing TCRζ was transfected into T cells by nucleofection. Phosphorylated TCRζ, phosphorylated NF-κB, and the IL-2 level in TCRζ-transfected CD3+T cells that were activated with anti-CD3 and anti-CD28 antibodies were measured by Western blot and enzyme-linked immunosorbent assay (ELISA). Significantly increased TCRζ levels were found in TCRζ-transfected CD3+T cells. After CD3 and CD28 antibody stimulation, a significantly higher phosphorylated TCRζ chain level was demonstrated, and an increased IL-2 production in TCRζ-upregulated T cells was associated with the increased expression of the phosphorylated NF-κB. In conclusion, TCRζ gene transfection could restore TCRζ chain deficiency and enhance IL-2 production in T cells from patients with CML. It is possible that TCRζ chain reconstitution in leukemia-specific, clonally expanded T cells will effectively increase their activation of antileukemia cytotoxicity.

Characterization of the CDR3 structure of the Vβ21 T cell clone in patients with P210BCR-ABL-positive chronic myeloid leukemia and B-cell acute lymphoblastic leukemia

The clonally expanded T cells identified in most cancer patients that respond to tumor-associated antigen such as P210(BCR-ABL) protein have definite, specific antitumor cytotoxicity. T cell receptor (TCR) Vβ CDR3 repertoire diversity was analyzed in patients with chronic myeloid leukemia (CML) and BCR-ABL(+) B-cell acute lymphoblastic leukemia (B-ALL) by GeneScan. A high frequency of oligoclonal expansion of the TCR Vβ21 subfamily was observed in the peripheral blood of CML and B-ALL patients. These clonally expanded Vβ21 T cells were correlated with the pathophysiologic process of CML. A conserved amino acid motif (SLxxV) was observed within the CDR3 region in only 3 patients with CML. Preferential usage of the Jβ segments was also observed in a minority of patients. The 3-dimensional structures of the CDR3 region containing the same motif or using the same Jβ segment displayed low similarity; on the contrary, the conformation of the CDR3 region containing no conserved motif in some T cell clones was highly similar. In conclusion, our findings indicate a high frequency of TCR Vβ21 subfamily expansion in p210(BCR-ABL)-positive CML and B-ALL patients. The characterization of the CDR3 structure was complex. Regrettably, at this time it was not possible to confirm that the Vβ21 T cell clones were derived from the stimulation of p210(BCR-ABL) protein.

Expression feature of CD3, FcɛRIγ, and Zap-70 in patients with chronic lymphocytic leukemia

Abstract In leukemia patients, T-cell function has been suppressed with the disease progress. Patients with chronic lymphocytic leukemia (CLL) are all to a degree immunodeficient. In order to elucidate the feature of T-cell receptor signal transduction in CLL, the expression levels of CD3γ, δ, ɛ, and ζ chain, FcɛRIγ, and Zap-70 genes in peripheral blood mononuclear cells (PBMCs) were analyzed. Real-time polymerase chain reaction with SYBR Green technique was used for detecting the gene expression level in PBMCs from 13 patients with CLL, 13 healthy individuals, and 10 B-cell acute lymphocytic leukemia (B-ALL) served as control. The β2-microglobulin gene was used as an endogenous reference. Relative mRNA expression level of genes was analyzed by using the 2−ΔCt × 100% method. Significant lower expression levels of CD3γ, ɛ, and ζ chain genes, as well as FcɛRIγ gene were found in CLL samples. Moreover, there was lost the negative correlation of the expression levels between CD3ζ and FcɛRIγ genes. The expression level of Zap-70 in CLL was lower than those from healthy controls, while higher than those from B-ALL group. There was no significant correlation between the expression levels of CD3ζ and Zap-70 genes neither in the healthy group nor in the CLL group. In conclusion, the results provide a global gene expression profile of CD3γ, δ, ɛ, and ζ chains, and the CD3ζ-related genes FcɛRIγ and Zap-70 in CLL. Deficiency of these gene expression levels might represent the feature related to T-cell immunodeficiency. The study might contribute to better understand the cellular immune features in CLL patients.

Evolution of T-cell clonality in a patient with Ph-negative acute lymphocytic leukemia occurring after interferon and imatinib therapy for Ph-positive chronic myeloid leukemia

IntroductionThe development of Philadelphia chromosome (Ph) negative acute leukemia/myelodysplastic syndrome (MDS) in patients with Ph-positive chronic myeloid leukemia (CML) is very rare. The features of restrictive usage and absence of partial T cell clones have been found in patients with CML. However, the T-cell clonal evolution of Ph-negative malignancies during treatment for CML is still unknown.ObjectiveTo investigate the dynamic change of clonal proliferation of T cell receptor (TCR) Vα and Vβ subfamilies in one CML patient who developed Ph-negative acute lymphoblastic leukemia (ALL) after interferon and imatinib therapy.MethodsThe peripheral blood mononuclear cells (PBMC) samples were collected at the 3 time points (diagnosis of Ph-positive chronic phase (CP) CML, developing Ph-negative ALL and post inductive chemotherapy (CT) for Ph-negative ALL, respectively). The CDR3 size of TCR Vα and Vβ repertoire were detected by RT-PCR. The PCR products were further analyzed by genescan to identify T cell clonality.ResultsThe CML patient who achieved complete cytogenetic remission (CCR) after 5 years of IFN-α therapy suddenly developed Ph-negative ALL 6 months following switch to imatinib therapy. The expression pattern and clonality of TCR Vα/Vβ T cells changed in different disease stages. The restrictive expression of Vα/Vβ subfamilies could be found in all three stages, and partial subfamily of T cells showed clonal proliferation. Additionally, there have been obvious differences in Vα/Vβ subfamily of T cells between the stages of Ph-positive CML-CP and Ph-negative ALL. The Vα10 and Vβ3 T cells evolved from oligoclonality to polyclonality, the Vβ13 T cells changed from bioclonality to polyclonality, when Ph-negative ALL developed.ConclusionsRestrictive usage and clonal proliferation of different Vα/Vβ subfamily T cells between the stages of Ph-positive CP and Ph-negative ALL were detected in one patient. These changes may play a role in Ph- negative leukemogenesis.

Enhancement of the TCRζ expression, polyclonal expansion, and activation of t cells from patients with acute myeloid leukemia after IL-2, IL-7, and IL-12 induction.

Defective T cell receptor (TCR) signaling resulting in lower T cell function plays a crucial role in the pathogenesis of T cell immunodeficiency in leukemia. Previous studies have indicated that lower TCRζ levels are a common characteristic of patients with leukemia, and upregulating TCRζ could partially recover T cell function. In this study, we characterized the effect of the stimulating factor induction on the TCRζ, Zap-70, and FcɛRIγ levels, IFN-γ secretion, and the distribution and clonal expansion of TCR Vβ subfamilies in CD3+ T cells sorted from peripheral blood from acute myeloid leukemia (AML) patients. The induction included single stimulating factor or a combination with different cytokines (IL-2, IL-7, IL-2+IL-7, IL-7+IL-12, CD3, CD3+CD28 antibody, CD3+CD28 antibody+IL-2, and CD3+CD28 antibody+IL-7) at 72 h. The results showed that increased TCRζ and Zap-70 levels with deceased FcɛRIγ in T cells after induction, and different responses to cytokine in T cell from different cases may indicate th...Defective T cell receptor (TCR) signaling resulting in lower T cell function plays a crucial role in the pathogenesis of T cell immunodeficiency in leukemia. Previous studies have indicated that lower TCRζ levels are a common characteristic of patients with leukemia, and upregulating TCRζ could partially recover T cell function. In this study, we characterized the effect of the stimulating factor induction on the TCRζ, Zap-70, and FcɛRIγ levels, IFN-γ secretion, and the distribution and clonal expansion of TCR Vβ subfamilies in CD3(+) T cells sorted from peripheral blood from acute myeloid leukemia (AML) patients. The induction included single stimulating factor or a combination with different cytokines (IL-2, IL-7, IL-2+IL-7, IL-7+IL-12, CD3, CD3+CD28 antibody, CD3+CD28 antibody+IL-2, and CD3+CD28 antibody+IL-7) at 72 h. The results showed that increased TCRζ and Zap-70 levels with deceased FcɛRIγ in T cells after induction, and different responses to cytokine in T cell from different cases may indicate the heterogeneity of T cells and different immune statuses in different AML cases. Increased IFN-γ levels in T cells from AML patients were detected after induction in the IL-12+IL-7, CD3+CD28+IL-2, and CD3+CD28+IL-7 groups. Moreover, the number of TCR Vβ subfamily T cells expressed was increased; however, all of the TCR Vβ subfamily T cells in the AML patients could not be completely recovered after induction. In conclusion, the cytotoxicity and activation function of T cells could be enhanced after induction by different stimuli accompanied by an increase in TCRζ and Zap-70 and recovery of the TCR Vβ repertoire in AML patients.

Upregulated TCRζ improves cytokine secretion in T cells from patients with AML

Previous studies indicated that upregulating TCRζ partially recovers T cell function in patients with leukemia. In this study, we characterized the cytokine profile of TCRζ-transfected T cells from acute myeloid leukemia (AML) patients by Quantibody®Array Glass Chip. Firstly, the significantly lower expression of TCRζ in CD3+/TCRζ+ cells from AML patients was found. Increased secretion of IL-2, IL-8, IL-10, IL-13, IFN-γ, TNF-α, GM-CSF, growth-regulated oncogene (GRO), MIP-1b, and regulated on activation, normal T cell expressed and secreted (RANTES) could be detected in T cells from AML patients after TCRζ upregulating. We concluded that upregulating TCRζ in T cells from AML can alter the secretion profile of cytokines and chemokine which are involved in T cell proliferation and activation.