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Objective: To evaluate the possible mechanism of transcription factors B cell lymphoma 6 (Bcl-6) , forkhead/winged helix transcription factor 3 (Foxp3) and retinoic acid related orphan receptor (RORγt) in CD4(+) T cells for immuno-related hematocytopenia (IRH)., Methods: CD4(+) T cells were harvested from 40 IRH patients, 38 aplastic anemia subjects and 25 normal controls and separated by magnetic activated cell sorting (MACS). Then the expressions of transcription factors of Foxp3, RORγ and Bcl-6 in CD4(+) T cells were measured by real time fluorescent quantitative-polymerase chain reaction (QRT-PCR)., Results: Auto-antibody was detected on CD34(+) cells (67.5% (27/40) ), CD15(+) cells (65.0% (26/40)), GlyA(+) cells (75.0% (30/40) ), auto-antibody involving three, two or one myeloid cell were detected in 27.5% (11/40), 52.5% (21/40), 20.0% (8/40) of IRH patients. Compensatory increase of Foxp3 mRNA was found in IRH (0.124 (0.073-0.198) vs 0.071 (0.046-0.118), P < 0.05). The expression of Bcl-6 was higher (2.243 (0.854-4.544) vs 1.211 (0.131-2.816), P < 0.05). Compared to aplastic anemia, the expression of RORγt was lower in IRH (0.133 (0.068-0.189) vs 0.290 (0.138-0.480), P < 0.01) and the ratio of Treg/Th17 shifted to Th17 in patients with aplastic anemia (Foxp3/RORγt ratio,0.500 (0.240-0.795) vs 0.975 (0.483-1.416), P < 0.01)., Conclusion: As one kind of bone marrow failures caused by autoantibody to bone marrow cells, IRH may occur due to a high expression of Bcl-6 in CD4(+) T cells, its immunopathogenesis is different from that of aplastic anemia.

Objective: To evaluate preliminarily the significance of detecting the Wilms' tumor (WT1) gene level on monitoring minimal residual disease (MRD) and predicting the clinical outcome in patients of acute leukemia following hematopoietic stem cell transplantation (HSCT)., Methods: The mRNA expression levels of WT1 and house-keeping gene ABL were dynamically measured with Real-time quantitative reverse transcription polymerase chain reaction (RQ-RT-PCR) on 326 bone marrow samples from 63 post-HSCT patients in our hospital from December 2001 to September 2009. After comparing the WT1 levels of patients with different post-transplantation outcomes, the investigators used the receiver operating characteristic (ROC) curve to determine the WT1 threshold so as to predict their clinical relapses. Then different prognoses of WT1 positive and negative patients were analyzed., Results: The levels of WT1 expression showed significant difference between the 19 relapsing and 44 non-relapsing patients with the median expression levels of 1270 (55 - 47 596) and 132 (0 - 2959) respectively (P < 0.01). In 19 relapsing patients, except for 1 patient discontinuing the detection of WT1, 10 mortality cases due to recurrence had higher levels of WT1 expression than other 8 patients (P > 0.05). According to the ROC curve, the cut-off value of WT1 at 585 could separate 63 patients into the WT1-positive group (> 585) and the WT1-negative group (≤ 585). The WT1-negative group was found to have a longer relapse-free survival (RFS) and overall survival (OS) than the positive group (all P < 0.01). Twenty-one WT1-positive patients were followed up for 3, 4 - 6, 7 - 9 and 9 months respectively. The cumulative post-HSCT recurrence rates in those WT1-positive cases were 8/8, 2/4, 2/4 and 3/5 (P = 0.063) respectively. And the intervention was ineffective., Conclusion: WTl gene may be an independent factor of monitoring MRD. And WT1 > 585 is a poor post-HSCT prognostic factor for the patients of acute leukemia.

Objective: To investigate the effects of targeting elimination of the leukemic CD34+ progenitor cells by using cytotoxic T lymphocytes (CTLs) specifically against Wilms tumor gene (WT1)-derived peptide., Methods: A 9-mer WT1 peptide (CMTWNQMNL) containing HLA-A * 0201-binding anchor motifs was synthesized. The suspended cells were collected from the culture of the peripheral blood mononuclear cells and divided into 2 groups: Group D (pure T cells) and Group C (IL2 + T cells). The dendritic cells (DCs) generated from the peripheral blood mononuclear cells of an HLA-A* 0201-positive healthy donor were repeatedly loaded with WT1 peptide so as to elicit cytotoxic T cells (CTLs) specifically for WT1 peptide, and restricted by HLA-A * 0201 (Group A). The specific lysis effects of WT1 peptide specific CTLs upon leukemic bone marrow CD34+ progenitor cells positively expressing WT1 (3 being HLA-A * 0201(+) and 3 being HLA-A*0201(-)), peripheral blood CD34+ cells from healthy persons (2 being HLA-A * 0201(+) and 1 being HLA-A * 0201(-)), and leukemia cells of the lines of NB4 (HLA-A * 0201(+)/WT1(+)), U937 (HLA-A * 0201(+)/WT1(-)), and K562 (HLA-A * 0201(-)/WT1(+)) were measured by methyl thiazolyl tetrazolium (MTT) chromatometry assay. The colony-forming unit-granulocyte macrophage (CFU-GM) forming capability of the leukemic bone marrow CD34+ progenitor cells and peripheral blood CD34+ cells from healthy persons treated with WT1 peptide specific CTLs were also determined by methylcellulose medium colony-forming unit assay. The CTLs elicited by DCs without WT1 peptide loading(Group B)and T cells cultured with IL-2 (Group C) were taken as controls., Results: The CTLs specific for WT1 peptide and restricted by HLA-A * 0201 (Group A) exerted a specific lysis upon the 3 cases with HLA-A * 0201(+)/WT1(+) leukemic bone marrow CD34+ progenitor cells and NB4 leukemia cells, the specific lysis levels were 55.3% +/- 2.8%, 67.1% +/- 3.2%, 49.4% +/- 3.8% and 55.0% +/- 3.7% respectively, all significantly higher than those upon the 3 cases with HLA-A * 0201(-)/WT1(+) leukemic bone marrow CD34+ progenitor cells, normal healthy peripheral blood CD34+ cells of the healthy persons, and leukemia cell of the lines K562 and U937 (the specific lysis levels were all < 20%). The specific lysis level of Group A CTLs was significantly higher than those of Group B and Group C CTLs (both P < 0.01). The relative colony formation rates of WT1-CTLs in Group A upon the 2 cases with HLA-A * 0201(+)/WT1(+) leukemic CD34+ progenitor cells were 17.8% +/- 4.0% and 20.8% +/- 3.41% respectively, both significantly lower than those of the none WT1-CTLs in Group B (88.9% +/- 3.4% and 91.8% +/- 5.7% respectively, both P < 0.01). There was no significant difference in the relative colony formation rate upon HLA-A * 0201(+)/WT1(-) leukemic bone marrow CD34+ progenitor cells and the HLA-A * 0201(-)/WT1(-) or HLA-A * 0201(+)/WT1(-) normal peripheral blood CD34+ progenitor cells between the CTLs of Groups A and B., Conclusions: The CTLs specific for WT1 and restricted by HLA-A * 0201 can exert specific lysis upon leukemic CD34+ progenitor cells highly expressing WT1 gene, and can inhibit the CFU-GM colony formation of such leukemic CD34+ progenitor cells. The product of expression of WT1 gene may be used as a target in the leukemic CD34+ cells.

Objective: To predict T-cell immune reconstitution by investigating T cell receptor excision circles (TREC) and T-cell receptor beta-chain variable region (TCRBV) clonal repertoire in leukemia patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT)., Methods: Real-time quantitative PCR was used to detect the TREC in 43 leukemia patients undergoing matched sibling donor bone marrow transplantation (MSD BMT), matched unrelated donor (MUD) BMT, or haploidentical-stem cell transplantation (HID-SCT), and in 70 normal individuals. RT-PCR was used to amplify 24 subfamily genes of T-cell receptor beta-chain variable region (TCRBV) in 24 of the 43 patients and 5 normal donors as control. The PCR products were further analyzed by genescane to evaluate the clonality of BV subfamily, characteristics of complementarity determining region 3 (CDR3), and the usage rate in BV subfamily., Results: There were (335.1 +/- 782.5) copies/10(5) cells in the 43 patients before transplantation, far lower than the normal value. The TREC values of the patients of the 3 groups all decreased obviously 3 months after transplantation. The TREC value of the MSD-BMT group recovered faster than the other two groups and reached the value before transplantation in 24 months. The recovery of TREC value in the HID-BMT group was delayed. 3 - 19 months after transplantation, the usage of TCRBV subfamilies was still restricted. There were 6 - 16 BV subfamilies expressed and 33% - 48% of them were polyclonals, the others were monoclones and oligoclones and existed in 24 BV subfamilies, no common monoclone BV subfamilies was expressed., Conclusion: Investigation of the TREC and TCRBV clonal repertoire showed that the number of naive T cell is lower and the usage of TCRBV subfamilies skewed 3 - 24 months after allo-HSCT. The immune deficiency of the patients undergoing HID-BMT is more prominent and consistent with the clinical process.

Objective: To investigate the functional role of human tissue inhibitor of metalloprotease (TIMP)-2 gene on the proliferation and infiltrating capability of human monocytic leukemic cells., Methods: Human monocytic leukemic cells of the line SHI-1 were cultured and the TIMP-2 expression on the cell membrane was detected by flow cytometry (FCM). The SHI-1 cells were transfected with human TIMP-2 gene (SHI-1/TIMP-2 cells) or blank vector (SHI-1/MSCV cells). The TIMP-2 expressed on the surface of the cell membranes of SHI-1/TIMP-2 and SHI-1/MSCV cells was detected by FCM. The SHI-1/TIMP-2 and SHI-1/MSCV cells were inoculated into the 96-well plate, 24, 48, 72, and 96 hours later MMT method and ELISA were used, and the cell growth curve was drawn to detect the proliferation ability of the cells. Matrigel was put into the upper layer of Transwell chamber. Human bone marrow matrix cells (BMMC) of leukemia patient and SHI-1/TIMP-2 cells or SHI-1/MSCV cells were put into the upper layers as experimental groups, and SHI-1/TIMP-2 cells or SHI-1/MSCV cells only, without human BMMC, were put into the upper layers as control groups. 72 hours later blood cell counting plate was used to measure the number of cells migrating through Matrigel. SHI-1/TIMP-2 cells or SHI-1/MSCV cells were injected intravenously into pre-treated BALB/c nu/nu mice. Thirty days later 8 mice from each group were killed to observe the tumorigenesis in the organs, especially the central nervous system leukemia (CNL). The survival of the other mice was observed., Results: The expression level of TIMP-2 on the cell membrane of the SHI-1/TIMP-2 cells was 99.3% +/- 0.1%, significantly higher than that of the SHI-1/MSCV cells (85.9% +/- 2.6%, P < 0.05). The A values 24, 48, 72, and 96 hours later of the SHI-1/TIMP-2 cells were 0.34 +/- 0.05, 0.6 +/- 0.05, 0.97 +/- 0.12, and 1.28 +/- 0.06 respectively, all significantly higher than those of the SHI-1/MSCV cells (0.28 +/- 0.03, 0.36 +/- 0.03, 0.54 +/- 0.09, and 0.99 +/- 0.03 respectively, all P < 0.05). The SHI-1/TIMP-2 cells and SHI-1/MSCV cells only could not migrate through Matrigel basically. 24 - 48 hours after co-cultivation Shi-1 cells began to appear in the lower layer of Transwell chambers, 72 hours later the trans-Matrigel SHI-1/TIMP-2 cells accounted for 24.7% +/- 6.9% of the inoculated SHI-1/TIMP-2 cells, a proportion significantly higher than that in the case of the SHI-1/MSCV cells (12% +/- 1.4%, P < 0.05). 24 days after the inoculation the mean body weight of the mice inoculated with SHI-1/TIMP-2 cells was 21.5 g +/- 0.4 g, significantly higher than that of the mice inoculated with SHI-1/MSCV cells (17.4 g +/- 0.6 g, P < 0.01). The mice inoculated with SHI-1/TIMP-2 cells showed much more tumors in different organs and much more severe infiltration in the CNS in comparison with the mice inoculated with SHI-1/MSCV cells The mean survival time of the mice inoculated with the SHI-1/TIMP-2 cells was 33.7 days, significantly shorter than that of the mice inoculated with SHI-1/MSCV cells (40 days)., Conclusion: TIMP-2 expressed on the cell membrane is critical to promote the proliferation and infiltration of SHI-1 cells.

Objective: To investigate the effect of targeting immunotherapy for leukemia cells by using cytotoxic T lymphocyte (CTL) specifically against WT (Wilm's tumor) 1-derived peptide., Methods: A 9-mer WT1 peptide (CMTWNQMNL) containing HLA-A*0201-binding anchor motifs was synthesized. Dendritic cells (DCs) generated from the peripheral blood mononuclear cells of an HLA-A*0201-positive healthy donor were cultured and divided into 2 groups: experimental group to be loaded with Wilms' tumor 1 (WT1) peptide so as to elicit CTL's specifically for WT1 peptide, restricted by HLA-A*0201, and control group. Six days later rhTNF-alpha was added for 3 days more to promote the maturation of DCs. Before loading of WT1 peptide and 2 days after the addition of rhTNF-alpha direct immunofluorescence labeling method was used with PE or FITC labeled mono-antibodies to detect the expression of the surface antigens: CD83, CD1alpha, CD80, CD86, CD14, and HLA-DR. DCs suspended and attached to wall were collected and then divided into 2 groups: pure T cell group (group D) to be cultured in culture medium without IL-2, and IL-2 + T cell group (Group C) to be cultured in 1640 culture medium with IL-2. Eight days later the T cells of Group C were co-cultured with the DCs of the experimental group (WTI peptide + DC + IL-2 + T cells, Group A) or the DCs of the control group (DC + IL-2 + T cells, Group B). Five days later the killing activity was detected. The CTLs of Groups A, B, and C at logarithmic growth phase were mixed with leukemic cells of the lines: NB4/WT1D, NB4WT1A, NB4 (all HLA-A*0201 +, WT1 +), U937 (HLA-A*0201 +, WTl -), and K562 (HLA-A*0201 -, WTI +), and mononuclear cells of the bone marrow of leukemic patients at different effector cell-target cell of 20:1 and 10:1. MTT method was used to examine the killing rate of CTL to the target cells., Results: (1) The killing rates of Group A cells to NB4/WT1 D, NB4WTA, and NB4, leukemic cells were 60.4% +/- 3.1%, 56.4% +/- 5.7%, and 55.0% +/- 3.7%, all significantly higher than those of the Group B cells (10.9% +/- 1.6%, 11.1% +/- 2.7%, and 11.9% +/- 2.5%), and those of Group C cells (9.1% +/- 1.0%, 9.2% +/- 1.7%, and 9.4% +/- 1.8%) (all P < 0.01). There were no significant differences in the killing rates to U937 and K562 leukemic cells among the 3 groups. (2) When the effect-target ratio was 20: 2, the killing activity of the CTLs of Group A to the HLA-A*0201 +, WT1 + NB4/WT1 D, NB4/WT1A and NB4 leukemic cells was significantly higher than those to the HLA-A*0201 +, WT1 - U937 cells and the HLA-A*0201 -, WT1 + K562 cells (both P < 0.001), however, not significantly different from that to the U937 and K562 cells. (3) There were no significant differences in the killing activity of Group A cells to NB4/WT1D, NB4/WT1A, and NB4 cells (P = 0. 065, P = 0.621). (4) When the effect-target ratio was 10:2, the killing rates of Group A cells to the NB4/ WT1D, NB4/WT1A, and NB4 cells were 45.9% +/- 3.9%, 43.9% +/- 3.7%, and 44.1% +/- 3.2% respectively, all significantly lower than those when the effect-target ratio was 20:1 (all P < 0.01)., Conclusion: CTLs specific for WT1 and restricted by HLA-A*0201 exert specific lysis upon leukemia cell lines and primary leukemia cells, but not upon normal hematopoietic cells, which provides a rationale for developing a strategy of WT1 peptide-based adoptive T-cell therapy and vaccination for human leukemia and solid tumors.

Objective: To investigate the significance of monitoring Wilms' tumor gene (WT1) expression level in bone marrow of leukemia patients following allogeneic bone marrow transplantation (allo-BMT)., Methods: Real-time quantitative reverse transcription polymerase chain reaction method was established for measuring WT1 and GAPDH expression levels in bone marrow cells of 15 patients with leukemia, including a total of 111 specimens during the follow-up, and in 23 non-leukemia patients by using LightCycler. Normalized WT1 expression level (WT1(N)) was determined as a ratio of WT1 to GAPDH times 10(4)., Results: The median expression levels of WT1(N) in 17 samples of newly diagnosed patients, 6 samples of relapsed patients, 88 samples from leukemia patient in complete remission and 23 samples of non-leukemic controls were 40.18 (5.48 to 510.27), 125.89 (34.50 to 273.95), 4.80 (0 to 56.96) and 1.47 (0 to 8.56) respectively. Nonparameter statistic analysis (Mann-Whitney U test) showed that the WT1(N) expression levels in the newly diagnosed group and relapsed group were statistically higher than in those in the complete remission group and non-leukemic controls (all P < 0.01), without significant differences between the complete remission group and control group (P = 0.692) and between the newly diagnosed group and relapsed group (P = 0.595). In general, the dynamic curves of WT1(N) levels following allo-BMT were consistent with the tendency of changes in expression levels of corresponding fusion genes for minimal residual disease (MRD) monitoring. Spearman Rho correlation analysis revealed that the correlation coefficient between the WT1(N) expression levels and BCR/ABL, AML/ETO, PML/RARalpha and MLL/AF17 fusion genes expression were 0.678 (P = 0.00), 0.677 (P = 0.00), 0.806 (P = 0.00) and 0.553 (P = 0.049) respectively. Three patients relapsed after allo-BMT and one patient relapsed before allo-BMT during the follow-up. A re-increment of WT1(N) expression level during follow-up could be detected 40 to 180 days earlier to hematological relapse., Conclusion: The WT1 expression level of leukemia patients following allo-BMT measured by real time RT-PCR can be a useful tool for monitoring MRD and warning the clinical relapse during follow-up.

Objective: To study the gene expression profiles of patients with myelodysplastic syndrome (MDS) and try to identify some genes with pathogenetic and diagnostic relevance., Methods: The bone marrow mononuclear cells (BMMNCs) of 10 patients with MDS, including 4 cases of refractory anemia (RA), 1 case of refractory thrombocytopenia (RTC), 4 cases of RA with excess blasts (RAEB), and 1 case of RAEB in transformation (RAEBt), were isolated and the total RNA was extracted and underwent transcriptome analysis by using customized cDNA microarray with 500 gene clones. Seventeen specimens of normal bone marrow, as controls, were collected from the ribs of 17 patients with chest tumors. Cluster software was used to make analysis. Semiquantitative RT-PCR was carried out to confirm the results of microarray analysis., Results: 95 genes, such as the genes of thrombospondin 1 (THBS1), phosphatase and tensin homolog (PTEN), MAX dimerization protein (MAD), DNA-damage-inducible transcript 3 (DDIT3), ets variant gene 1 (ETV1), G1 to S phase transition 1 (GSPT1), were shown to be abnormally expressed in at least 5 MDS patients compared to the normal controls, involving cell growth and differentiation regulation, cell cycle control, signaling, and redox. The 10 MDS patients in different stages were clustered into two distinct groups, whereas a case with refractory thrombocytopenia and other RA patients were clustered into two subgroups. Semiquantitative RT-PCR revealed the identical results in 3 (60%) of the 5 genes determined by microarray analysis. Further analysis on samples from 50 MDS patients confirmed the different expression levels of RNA helicase-related protein (RNAHP), GSPT1, and DDIT3 between the MDS patients and the normal controls., Conclusion: The technology of microarray can reveal the intrinsic molecular features of MDS patients and the detection of DDIT3 and RNAHP expression may be useful for the diagnosis of MDS.