Screening and evaluation of Smo-siRNA targeted to inhibition of Molt-4 cell proliferation / 中国组织工程研究

BACKGROUND: Studies have shown that the occurrence and development of T lymphocytic leukemia is related to the abnormality of Hedgehog pathway. The Smo gene is a key gene in this signaling pathway and controls the transmission of Hedgehog signaling into the cell membrane. OBJECTIVE: To design and screen a highly efficient and specific Smo-siRNA which is able to downregulate the Smo gene expression in Molt-4 cells, thereby inhibiting the Molt-4 cells proliferation and inducing apoptosis. METHODS: (1) Smo-siRNAs numbered 1, 2 or 3, and the scrambled non-siRNA control (SC) were obtained by chemosynthesis. Untreated and sc-treated cells were used as controls. (2) Smo expression levels in Molt-4 cells were analyzed using qRT-PCR at 24, 48, 72 hours after siRNAs delivered by NuclefectorTM.Cell proliferation in vitro was assayed by the cell counting kit-8.The morphology and percentage of apoptotic cells were revealed by Hoechst33258 staining and flow cytometry, respectively. RESULTS AND CONCLUSION: (1) Smo-siRNAs were successfully transferred into Molt-4 cells, and exhibited best silencing results. After transfection with Smo-siRNA1, the mRNA level of Smo was significantly reduced (P < 0.05), and the lowest level was at 48 hours after transfection. (2) Cell proliferation of Molt-4 cells was significantly inhibited by Smo-siRNA at 24 hours after transfection. (3) Hoechst staining results showed morphological changes of Molt-4 were in accordance with those of apoptotic cells. (4) The apoptotic rate was significantly increased in the Smo-siRNA group compared with the control group (P < 0.05). Findings from this study showed that suppression of Smo by RNA interference could effectively inhibit proliferation and induce apoptosis in Molt-4 cells, indicating that Smo-siRNA as gene targeted therapy or synergistic treatment has therapeutic potential in T-cell malignancies.

Effects of Down-regulating PPP2R5C Expression on Expression Profile of TAL1-related Regulating Genes in Jurkat Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>Based on our previous study showing the inhibition of lenkemia T cell proliferation by down-regulating PPP2R5C expression, this study was aimed to analyze the influence of down-regulating PPP2R5 expression via RNA interference on genes relatied with TAL1 signaling pathway by using gene chip technique.</p><p><b>METHODS</b>The PPP2R5C-siRNA799 was transduced into Jurkat cells by nucleofection, the total RNA was isolated from treated Jurkat cells after culture for 48 hours; the target sequences were prepared by revevse transcription after mRNA purification, and were hybridized with affymetrix gene expression profile chip 3' IVT. The original image data were collected using affymetrix gene chip scanner 3 000, and the gene expression profile was analyzed using gene spring GX 11.0 soflware.</p><p><b>RESULTS</b>The expression of all 26 genes related with TAL1 signaling pathway was changed, out of which the expression of 15 genes were up-regulated and the expression of 11 genes was down-regulated in PPP2R5C-siRNA 799-transfected Jurkat cells. The genes with significantly up-regulated expression were GATA1, TCF4, XRCC6 and TCF3, while the genes with significantly down-regulated expression were SIN3A and RUNX1.</p><p><b>CONCLUSION</b>The down-regulation of PPP2R5C gene expression in Jurkat cells via RNA interference to a certain degree can inhibit TAL1 signaling pathway genes, thereby suppresses the proliferation of Jurkat cells.</p>

One-year continuous observation of change in peripheral T cell subsets in workers exposed to low levels of benzene / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe the T cell subsets and blood cells in the peripheral blood of workers exposed to low levels of benzene for one year, and to investigate the relationship between T cell function impairment and benzene-induced hematopoietic injury after benzene exposure.</p><p><b>METHODS</b>Eighty-eight workers (58 males and 30 females, aged 18 ∼ 22 years) who just began to work in the workshop of a paint factory with exposure to benzene in Guangzhou, China were assigned to experimental group, and 88 workers (58 males and 30 females, aged 18 ∼ 25 years) who worked in the workshop without exposure to benzene were selected as controls. The blood samples of the workers were examined once every 4 months to measure the percentages of peripheral T cell subsets and peripheral blood cell counts in the one-year study. The benzene concentrations at operation points were also measured.</p><p><b>RESULTS</b>The peripheral blood cell counts in the benzene-exposed workers had no significant changes in the first and second examinations; the white blood cell (WBC) counts in the experimental group in the third and fourth examinations were significantly lower than that in the control group [(6.4 ± 3.0)×10(9)/L and (6.3 ± 2.7)×10(9)/L vs (7.3 ± 3.0)×10(9)/L, P < 0.05], and the platelet (PLT) count in the experimental group in the fourth examination was also significantly lower than that in the control group[(179 ± 74)×10(9)/L vs (189 ± 70)×10(9)/L, P < 0.05]. Compared with those in the control group (CD4+: 54.29 ± 12.78%, CD8+: 37.25 ± 12.30%), the percentage of CD3+ T cells in the experimental group increased in the third examination; the percentage of CD4+ T cells in the experimental group decreased continuously in the second, third, and fourth examinations (50.77 ± 11.05%, 45.40 ± 9.41%, and 41.27 ± 10.62%), while the percentage of CD8+ T cells in the experimental group kept increasing (46.07 ± 10.18%, 50.36 ± 10.62%, and 56.40 ± 9.41%) (P < 0.05).</p><p><b>CONCLUSION</b>The change in T cell subsets precedes that in the blood system in the workers exposed to low levels of benzene.</p>

Establishment of stable subline of K562 cells expressing human leucocyte antigen a1101 / 中国实验血液学杂志

The aim of this study was to establish a stable subline of K562 cells expressing the HLA-A(*)1101 protein, which was expected to provide target cells for characterizing the HLA-I restrictive antigen specific cytotoxic T lymphocyte (CTL) effects against chronic myeloid leukemia (CML). The HLA-A(*)1101 protein encoding gene was amplified from peripheral blood mononuclear cell (PBMNC) of CML patient by RT-PCR; the 2A peptide linker (D-V-E-X-N-P-G-P) gene was linked to the 3'terminal of the HLA-A(*)1101 gene by recombinant PCR, then the recombinant was cloned into the pEGFP-N3 plasmid which contains an enhanced green fluorescent protein gene, and the eukaryotic recombinant expression vector containing HLA-A(*)1101-T2A-EGFP transcription box was constructed; the pEGFP-N3 vector and recombinant vector was separately electroporated into K562 cells. The expression of GFP was monitored by fluorescence microscopy, finally stably transfected sublines of K562 cells containing HLA-A(*)1101 gene, and of K562 containing pEGFP-N3 vector were obtained by G418 selection; the transcriptional or translational expression of HLA-A(*)1101 gene was detected with RT-PCR and flow cytometry respectively. The results indicated that the eukaryotic expression vector HLA-A(*)1101-T2A-EGFP plasmid was successfully constructed; after G418 selection for 2 months, two sublines of K562 cells (HLA-A(*)1101(+)K562, pEGFP-N3(+)K562) expressing GFP were constructed. The expression of HLA-A*A1101 gene could be determined in HLA-A(*)1101(+)K562 cell line by RT-PCR, while the pEGFP-N3(+)K562 cells could not express HLA-A*A1101 gene. HLA-A(*)1101 protein and GFP double positive HLA-A(*)1101(+)K562 cells were up to 88.5%, which was obviously higher than pEGFP-N3(+)K562 cells (0.698%) by flow cytometric analysis. It is concluded that a simple and effective method to select HLA-A(*)1101(+)K562 cells has been established and a subline of K562 cell expressing HLA-A(*)1101 protein on its cell membrane was successfully constructed, which provides the tool cells for further studying the specific cellular immunity against-CML.

Change of expression pattern of CD3 genes in peripheral blood T-cells from CML patients / 中国实验血液学杂志

Our previous finding showed that down-regulation of CD3ζ gene was detected in patients with chronic myeloid leukemia (CML). In order to further elucidate the feature of T cell immune status in the signal transduction in CML patients, the expression patterns of all 4 CD3 genes were characterized in peripheral blood of patients, the expression levels of CD3γ, δ, ε and ζ chain genes were detected by real time qPCR with SYBR Green I staining in peripheral blood mononuclear cells (PBMNCs) from 17 cases of de novo CML patients in chronic phase and 17 cases of healthy individuals, the ß₂-microglobulin gene was used as an internal reference, and the mRNA expression level of each CD3 gene was evaluated by the 2(-ΔCt) x 100% method. The results showed that the median expression levels of CD3γ, δ and ε genes (2.344%, 0.515% and 3.516%) in CML patients were not significantly different from healthy individuals (p = 0.072, p = 0.190, p = 0.615, respectively), while the expression level of CD3ζ gene in PBMNCs from CML patients (0.395%) was lower than that from healthy individuals (1.538%) (p < 0.001). The expression patterns of 4 CD3 genes in proper order were CD3ε > CD3γ > CD3δ > CD3ζ in CML group, in contrast, the expression patterns were presented as CD3γ > CD3ε > CD3ζ > CD3δ in healthy group. It is concluded that the present study characterized the expression pattern of CD3γ, δ, ε and ζ chain genes in CML patients, lower expression of CD3ζ is the feature of TCR signal transduction immunodeficiency and the expression patterns of 4 CD3 genes are changed in CML patients.

Structural feature and biological function of PPP2R5C gene / 中国实验血液学杂志

PPP2R5C is one of the members of regulatory subunits of protein phosphatase 2A (PP2A), which plays a critical role in cell proliferation, differentiation and transformation, based on its induction of dephosphorylation of P53 at various residues. Recently, it was characterized that the alteration of expression pattern of PPP2R5C is associated with cell malignant transformation, thus PPP2R5C was thought as a marker for progressive disease in B-CLL. In this article the gene structure and biological function of PPP2R5C as well as relation of PPP2R5C with genesis and development of cancer were discussed.

Thymic recent output function in patients with B-cell lymphocytic malignancies / 中国实验血液学杂志

The aim of the study was to analyze the naive T cell level of thymic recent output in patients with B-cell malignancies, thereby to evaluate the potential T-cell function. Quantitative analysis of T-cell receptor rearrangement excision circles (TRECs) in DNA of peripheral blood mononuclear cells from 61 cases of B-cell lymphocytic malignancy (including 20 cases of adult B-ALL, 6 case of childhood B-ALL, 4 cases of B-CLL, 17 cases of B-NHL and 14 cases of MM) were preformed by real-time PCR (TaqMan), and TREC-level was detected according to the number of CD3-positive cells. 5 case of ALL-CR and 17 normal individuals were served as controls. The results showed a dramatic reduction of TREC values in all groups of patients. The mean value of TRECs was 0.53 +/- 1.52 copies/1000 PBMNC and 2.01 +/- 3.93 copies/1000 CD3+ cells in adult B-ALL (p = 0.0005, p = 0.0123), 0.11 +/- 0.15 copies/1000 PBMNC and 0.23 +/- 0.27 copies/1000 CD3+ cells in B-CLL (p = 0.0015, p = 0.0381), 0.71 +/- 1.34 copies/1000 PBMNC in B-NHL (p = 0.0017), 0.53 +/- 0.90 copies/1000 PBMNC in MM patients (p = 0.0018), as compared with 3.76 +/- 3.42 copies/1000 PBMNC and 5.87 +/- 4.96 copies/1000 CD3+ cells in normal individuals, the TREC level was significantly decreased in all groups of B-cell lymphocytic malignancy, as well as in ALL-CR group. However, the TREC level in childhood B-ALL was significant higher than those in adult B-ALL group. It is concluded that the function of thymic recent outputting naive T cells in B-cell malignancies significantly decreases, however, the individual difference of thymic output function is obvious. The thymic recent output function can not be recovered during CR phase in patients with B-cell malignancies, so that dynamic analysis of TREC level is necessary.

Effects of lead exposure on thymic output naive T cells function / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the levels of T cell receptor rearrangement excision DNA circles (TRECs) within peripheral blood from workers exposed to lead, and thereby to evaluate the number of naive T cells and recent thymic output function.</p><p><b>METHODS</b>Quantitative detection of TRECs in peripheral blood mononuclear cells (PBMNC) from 10 cases of workers exposed to lead was performed by real time PCR analysis. 11 workers without exposure to lead served as unexposed controls. In addition, the relationship between TRECs, age, length of service, blood lead, urea lead, blood ZPP and urea delta-ALA was investigated.</p><p><b>RESULTS</b>The mean value of TRECs in workers exposed to lead was (2.44 +/- 1.87)/1000 PBMC, significantly under (5.60 +/- 3.96)/1000 PBMC in unexposed controls. A significant negative correlation was found between the TRECs and urea-ALA. But there was no significant correlation between them after controlling for blood lead, urea lead.</p><p><b>CONCLUSION</b>Lead exposure may damage thymic output naive T cells function. Furthermore, low-level exposure to lead may damage immune system and earlier than expected.</p>

Changes of T lymphocyte subsets in workers with long-term benzene exposure / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe the changes of T-lymphocyte subsets in workers with long-term benzene exposure, and further understand the benzene's lymphotoxicity.</p><p><b>METHODS</b>Blood was sampled from 44 patients with chronic benzene poisoning of different degrees, (mild 22 patients, moderate 14, severe 8) respectively. Twenty-two health benzene exposed workers, and 94 health unexposed workers served as normal control. A total of the phenotype (CD4, CD8) of T lymphocyte in peripheral blood was analyzed by indirect immunofluorescence assay.</p><p><b>RESULTS</b>Lymphocyte subset analysis showed significantly decreased CD4(+) T lymphocytes, CD4(+)/CD8(+) ratio, except CD8(+) T lymphocytes in benzene exposed groups (P<0.05). Among the four benzene-exposed groups, CD4(+) T lymphocytes and CD4(+)/CD8(+) ratio showed no difference (P>0.05).</p><p><b>CONCLUSION</b>The primary changes of T-lymphocyte subsets in workers following benzene long-term exposure are the decrease of CD4(+)%, but the changes are not correlated with haematopoietic injury.</p>

T-cell receptor V alpha gene repertoire and clonal expansion in benzene-exposed workers / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe the distribution of TCR V alpha gene repertoire and clonal expansion in peripheral blood mononuclear cells from 9 donors and 16 workers exposed to benzene.</p><p><b>METHODS</b>Complementarity determining region 3 (CDR3) of TCR V alpha subfamily genes were amplified using RT-PCR. The PCR products were further analyzed by genescan to evaluate clonality of T cells.</p><p><b>RESULTS</b>Almost all of 29 V alpha subfamily could be detected in 9 donors. 1 approximately 11 V alpha subfamilies were identified in all but one of the workers studied. The most frequently expressed V alpha subfamily were V alpha 3, V alpha 12 and V alpha 19 (68.8%), V alpha 14 (56.3%), with a lower expression rate found in V alpha 5, V alpha 15, V alpha 16, V alpha 22, V alpha 23 and V alpha 24 (6.3%). Clonal expansion T cells in one or more V alpha subfamily were found in 12 out of all workers studied, including oligoclonal, oligoclonal trend and biclonal patterns. The frequency of clonal expansion T cells in V alpha 12, V alpha 14 and V alpha 19 subfamilies were higher than others.</p><p><b>CONCLUSION</b>Skewed distribution and clonal expansion of TCR V alpha subfamily T cells could be found in workers exposed to benzene. V alpha 12, V alpha 14 and V alpha 19 subfamilies may be highly sensitive to benzene exposed. This is the first report of clonal expansion TCR V alpha T cells in the benzene-exposed group. The bias pattern of TCR V alpha T cells may be due to the immune cytotoxicity from benzene. However, whether the oligoclonality in some V alpha subfamilies reflect the phenomenon of clone absence or may be a response clone to benzene-related impairment during exposed to benzene, remains an open question.</p>

Relationship between thymus output function in CML patients and their bcr-abl mRNA levels / 中国实验血液学杂志

The study was purposed to analyze the relationship between the content of T-cell receptor excision DNA circles (TREC) and bcr-abl mRNA levels in CML patients and to evaluate the prognostic significance of recent thymic output function detection in patients with chronic myelogenous leukemia (CML). Quantitative detection of TREC and bcr-abl fusion gene transcripts in peripheral blood from 15 CML patients were preformed by real-time PCR. The change of bcr-abl levels in 6 patients was followed-up for two years. The results showed that there was no significant correlation between TREC and bcr-abl mRNA levels in peripheral blood from CML patients for the first attack. Patients who had higher TREC at diagnosis had a larger reduction of bcr-abl after 2 years of follow-up. While out of 2 patients who underwent haemopoietic stem cell transplantation (HSCT), one patient with higher level of TREC before transplantation was confirmed to express undetectable level of TREC by three consecutive detections after transplantation, other one patient was identified to express low level of bcr-abl. It is concluded that high thymic output function in CML patients can be beneficial for killing the residual CML cells.

Peripheral blood naive T cell level and its T cell receptor Vbeta repertoire usage profile in patients with chronic myelogenous leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To analyze peripheral blood naive T cell level, its T cell receptor (TCR) Vbeta repertoire usage profile and clonality for evaluating the recent thymic output function and the expansion feature of TCR Vbeta subfamily T cells in patients with chronic myelogenous leukemia (CML).</p><p><b>METHODS</b>Quantitative detection of T-cell receptor excision DNA circles (TRECs) in peripheral blood mononuclear cells (PBMNC) from 20 cases of CML was preformed by real-time PCR (TaqMan) analysis, and TRECs-number in T-cells was calculated from peripheral blood CD3-positive cell rate. The expression and clonality analysis were detected by RT-PCR and Genescan technique in PBMNC from 14 out of the 20 patients. Nine normal individuals served as controls.</p><p><b>RESULTS</b>A dramatic reduction of TRECs value in patients with CML was detected as compared with that in normal controls. The mean value of TRECs was 0.06 +/- 0.16 copy/1000 CD3(+) cells in CML patients while 6.84 +/- 4.71 copies/1000 CD3(+) cells in normal controls (P < 0.01). The 1 - 12 Vbeta subfamilies were variably expressed in samples from 14 patients. Genescan analysis identified clonal expanded T cells of some Vbeta subfamily from 13 cases. Vbeta3, Vbeta10, Vbeta19, Vbeta21 and Vbeta22 subfamilies clonal T cells were more frequently seen.</p><p><b>CONCLUSION</b>There is a prominent reduction of recent thymic output naive T cells function in CML. The predominant usage and clonal expansion of TCR Vbeta subfamily T cells could be identified, indicating that CML patients have specific immune response to leukemia associated antigen, in spite of their T cell immunodeficiency.</p>

Expressions of transcription factor GATA-1 and GATA-2 genes in bone marrow stromal cells from patients with leukemia / 中国实验血液学杂志

In order to investigate expressions of transcription factor GATA-1 and GATA-2 genes in the bone marrow stromal cells (BMSCs) from patients with leukemia or normal controls, bone marrow stromal cells from 34 normal cases and 42 cases with leukemia were cultured long-term in vitro. Nonadherent cells (bone marrow hematopoietic cells) and amplified adherent cells (BMSC) were collected separately. Expressions of GATA-1 and GATA-2 genes were analyzed by using RT-PCR-ELISA; the semi-quantitative expression levels of GATA genes in the BMSCs from patients with leukemia were compared with normal controls. The results showed that expressions of GATA-1 and GATA-2 genes could be detected in the BMSCs and the bone marrow hematopoietic cells from both normal controls and the cases of leukemia. The expression ratio of GATA-1 in the BMSCs from acute lymphocytic leukemia (ALL) (85.7%) was similar to the normal controls (88.2%), whereas the expression ratios in BMSCs from acute myelocytic leukemia (AML) (55.6%) and chronic myelocytic leukemia (CML) (41.2%) were significant lower than the normal controls (P < 0.05). The rank of expression level of GATA-1 gene in the BMSCs was "ALL>AML>normal>CML". There was no difference in the expression level of GATA-2 gene within the BMSCs from normal controls and patients with leukemia. The ranks of expression levels of GATA-1 and GATA-2 genes in bone marrow hematopoietic cells were "AML>normal>ALL>CML" and "AML>CML>ALL>normal". The dominant expression of GATA-2 gene was found in the BMSCs from AML, CML or normal controls. It is inferred that the expressions of GATA-1 and GATA-2 genes in the BMSCs of normal controls and patients with leukemia may influence the regulation of hematopoiesis in the bone marrow stroma and it is worthy of further study to explore their roles in pathogenesis and development of leukemia.

Significant decrease of recent thymic output function in patients with severe benzene intoxication / 中国实验血液学杂志

The aim of the present study was to investigate the level of T-cell receptor excision DNA circles (TREC) in peripheral blood mononuclear cells (PBMNC) of patients with severe benzene poison, thereby to evaluate the content of naive T cells and the recent thymic output function. Quantitative detection of TREC in DNA of PBMNCs from 16 normal individuals and 8 cases with severe benzene poison was preformed by real-time PCR using TaqMan technique. The results showed that TREC level was 6.69 +/- 4.79/1 000 PBMNCs in normal individuals, however, significant decrease was shown in patients with severe benzene poison (1.03 +/- 0.44/1 000 PBMNCs, P < 0.01). The TREC level was persistently low in period of benzene poisoning, even if peripheral blood cell counts were at normal levels. In conclusion, the recent thymic output function is remarkably decreased in patients with severe benzene intoxication, that may obviously damage the T cell immune function.

Expression of SCL gene in bone marrow stromal cells from patients with leukemia / 中国实验血液学杂志

In order to investigate expression of SCL (stem cell leukemia) gene in bone marrow stromal cells (BMSC) and bone marrow cells from patients with leukemia and normal individuals, bone marrow mononuclear cells from AML (18 cases), CML (17 cases), ALL (7 cases) and normal individuals (33 cases) were cultured long-term in vitro. Nonadherent cells (hematopoietic cells) and amplified adherent cells (BMSC) were collected respectively. RT-PCR-ELISA assay was then performed to detect expression of SCL gene. The expression ratio of SCL gene were analyzed and its expression level was normalized by beta(2)M gene acting as an internal reference for the purpose of semi-quantitative analysis. The results indicated that the expression ratio of SCL gene was lower in BMSC from AML (27.8%) and CML (11.8%) than that in normal controls (69.7%, P < 0.05), while was higher in the nonadherent cells from CML (64.3%) than that in its corresponding BMSC (P < 0.05). Semi-quantitative analysis showed that SCL gene expression level in nonadherent cells from AML was higher than that in its corresponding BMSC (P < 0.05). In conclusion, the low-level expression state of SCL gene in BMSC from patients with AML and CML may be involved in the abnormal regulation of hematopoiesis in myelocytic leukemia.

Evaluation of recent thymic output function in benzene-exposed workers / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To analyze the content of signal joint T-cell receptor excision DNA circles signal joint T-cell receptor excision DNA circles (sjTRECs) within peripheral blood mononuclear cells (PBMCs), thereby to infer the level of naive T cells and the recent thymic output function in benzene-exposed workers.</p><p><b>METHODS</b>Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 11 normal individuals and 62 benzene-exposed workers were performed by real-time polymerase chain reaction (PCR) and TaqMan technique.</p><p><b>RESULTS</b>The median value of sjTRECs copies/1,000 PBMCs was 7.81 in normal individuals whereas it was 2.56 copies/1 000 PBMCs in age-unadjusted benzene-exposed workers (P < 0.01). And its levels were obviously different between two different age groups: that in 30-year-old group (1.76 copies/1,000 PBMCs, n = 23) was less than <or= 30-years-old group (3.19 copies/1,000 PBMCs, n = 39, P < 0.05).</p><p><b>CONCLUSIONS</b>Benzene exposure may influence the recent thymic output function, and the sjTRECs levels may relate with the age of benzene-exposed workers.</p>

Changes in the T-cell receptor V beta gene repertoire after allogeneic hematopoietic stem cell transplantation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>We distinguished graft-versus-host disease (GVHD) from graft-versus-leukemia (GVL) effects and to investigate the distribution of T-cell receptor (TCR) V beta gene repertoire in individuals with leukemia before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT).</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMC) were obtained from 10 normal individuals, 8 donors and 11 patients with leukemia before and after transplantation. Polymerase chain reaction (PCR) amplification of complementarity-determining region 3 (CDR3) of 24 TCR V beta genes was used to examine serial samples of PBMC. The PCR products were further analyzed by genescan to evaluate clonality of T cells.</p><p><b>RESULTS</b>The 24 TCR V beta gene repertoire displayed highly diverse and polyclonal spectratypes in all normal individuals and 4 of 8 donors. Another 4 donors expressed part of the 24 TCR V beta subfamily and 1 donor had oligoclonality. The expressions of the 24 TCR V beta subfamilies were skewed and restricted in 11 leukemia patients before and after transplantation. Some absences of 24 TCR V beta subfamily expression were quite similar between the recipients pro-transplantation and related donors. The number of subfamilies expressed increased over time post-transplantation, but the restricted expressions of the subfamily could last 6 - 30 months after transplantation. All patients with GVHD and some without GVHD exhibited T cell clonal expansion. The expansive T cell clone was distributed in V beta 2-3, 16-17, 18-19, 21 and V beta 23 in patients with GVHD and in V beta 7, 9, 16 and 19 in patients without GVHD. One patient with syngeneic-HSCT (syn-HSCT) had V beta 15 and 16 T cell expansion after transplantation. One patient displayed V beta 18 T cell expansion after donor lymphocyte infusion (DLI).</p><p><b>CONCLUSIONS</b>Normal individuals express the entire 24 TCR V beta gene repertoire and have polyclonal distribution. However, the TCR V beta gene repertoire is only partially expressed in some donors. The TCR V beta gene repertoire is restrictedly expressed in a skew fashion in patients with leukemia before and after transplantation. The number of TCR V beta gene subfamilies increases over time post-transplantation. GVHD and GVL effects may induce the proliferation of T cell clones. Clinical GVL response may be distinguished from GVHD alloreactivity through the host MHC antigen.</p>

Clonal expansion of TCR Vbeta subfamily T cells induced by bcr3-abl2 peptide / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the clonal expansion of T cell receptor (TCR) Vbeta subfamily T cells from cord blood induced by bcr3-abl2 peptide in vitro.</p><p><b>METHODS</b>T cells from 3 units of cord blood were amplified by anti-CD(3) monoclonal antibody (McAb) and IL-2 with or without synthetic b3a2 peptide. T cell specific cytotoxicity was analyzed by lactate dehydrogenase (LDH) assay, TCR Vbeta subfamilies by using reverse transcriptase-polymerase chain reaction (RT-PCR) and genescan technique.</p><p><b>RESULTS</b>bcr3-abl2 peptide specific cytotoxicity T cells were successfully induced from the 3 units of cord blood by synthetic b3a2 peptide. Compared with that in CD(3) McAb induced cells, distribution pattern of TCR Vbeta repertoire was different in T cells induced with b3a2 peptide. Oligoclonal and oligoclonal tendency TCR Vbeta subfamily T cells could be identified in cord blood T cells induced by b3a2 peptide in 1 or 2 weeks, whereas those induced by anti-CD(3) McAb and IL-2 were mostly polyclonal.</p><p><b>CONCLUSION</b>The cytotoxicity T cells with anti-CML specificity could be induced by b3a2 peptide. The specific anti-CML cytotoxicity may be derived from the clonal expansion TCR Vbeta subfamily T cells.</p>

T cell receptor Vbeta repertoire usage and clonal expansion of T cells in chronic myelogenous leukemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>In general, it is very important to understand the state of T cell immune response against tumor cells in leukemia patients and it is especially critical to assess the T cell repertoire of untreated patients. As we know, few studies have dealt with the distribution of oligoclonal T cells in leukemia, so we investigated the distribution and clonality of TCR Vbeta repertoire of T cells in patients with chronic myelogenous leukemia (CML) in chronic phase.</p><p><b>METHODS</b>The complementarity determining region 3 (CDR3) of TCR Vbeta24 subfamily genes were amplified in peripheral blood mononuclear cells from 27 cases with CML using reverse transcription-polymerase chain reaction (RT-PCR). In order to observe the distribution of TCR Vbeta repertoire, the PCR products were further analyzed by genescan technique to evaluate clonality of the detectable TCR Vbeta T cells. The PCR products of the oligoclonal T cells from three cases were analyzed by direct sequencing to define the sequence of CDR3.</p><p><b>RESULTS</b>The expression pattern of TCR Vbeta repertoire in different individuals are different. Vbeta2-21 subfamilies could be detected in CML cases. The frequent usage Vbeta repertoire in CML was Vbeta1, Vbeta2 or Vbeta13. Most of the PCR products from 27 patients displayed polyclonality, while a part of the PCR products from 21 out of 27 samples displayed clonal expansion pattern. The clonal expanded T cells in CML could be found in Vbeta16 subfamilies. The frequent usage of Vbeta genes in clonal expansion was Vbeta3, Vbeta13 or Vbeta21. Multiple Vbeta clonal expansion was a general phenomenon in the same patient. The CDR3 sequence of Vbeta21 oligoclonal T cells from 3 cases showed some difference in splice regions and in the usage of J segments.</p><p><b>CONCLUSIONS</b>These results indicated that clonal expanded T cells could be found in patients with CML and were tendentious in Vbeta3, Vbeta13 and Vbeta21 subfamilies that may be related to the specific immune response for leukemia cell associated antigen.</p>

Comparison of the clonal expansion of TCR Vbeta T cells in patients with acute promyelocytic leukemia in vivo and in vitro / 中国实验血液学杂志

In order to analyze the distribution and clonal expansion of TCR Vbeta subfamily T cells in patients with acute promyelocytic leukemia (APL) in vivo and in vitro after T cell culture, the peripheral blood mononuclear cells from 3 APL patients were expanded by rhIL-2 and anti-CD3 antibody using liquid T lymphocytes culture technique. The complementary determining region 3 (CDR3) of TCR beta with variable region genes was amplified in T cells from 3 APL cases before and after T cell culture by using RT-PCR. The positive products were further analyzed to identify the clonality of T cells by genescan. The results showed that only a part of 24 Vbeta subfamilies was detected in T cells from the patients, and some Vbeta subfamily T cells could be identified after T cells culture. The clonal expansion T cells in some TCR Vbeta subfamilies could be found in all patients. The similar oligoclonal expansion of Vbeta1, Vbeta3, Vbeta7, Vbeta16 and Vbeta20 T cells was detected in two cases at different time points after T cell culture. It is concluded that the restricted expression of TCR Vbeta subfamily in T cells from patients might be the common feature in leukemia. Some Vbeta subfamily T cells could be induced after T cells culture in vitro. The continual clonal expansion of TCR Vbeta subfamily T cells at different time points after T cells culture could be a specific immune response of patients T cells related to the specific APL cell associated antigen.