利用T细胞受体变异β基因谱系分析T细胞急性淋巴细胞白血病病人T细胞克隆性

中国病理生理杂志 2000年第7期第16卷 论著

作者：李杨秋　陈少华　杨力建　祁明芳

单位：(广州暨南大学医学院血液病研究室， 广东 广州 510632)

关键词：受体，抗原，T细胞；基因；白血病

　　［摘　要］　目的：分析T细胞-急性淋巴细胞白血病(T-ALL)病人的T细胞克隆性。方法：利用RT-PCR方法分析6例T-ALL和10例正常人外周血单个核细胞中24个T细胞受体变异β(TCR Vβ)基因的CDR3长度，PCR产物再进一步进行基因扫描和序列分析。结果：3例病人的某些TCR Vβ亚家族T细胞呈单克隆或寡克隆性增殖，主要为Vβ2、3、6、9、21和24。其它3例及正常人均表现为多克隆性增殖T细胞。结论：部分T-ALL来自于TCR Vβ亚家族克隆性增殖T细胞。该方法有助于临床上检测微小残留病变。

　　［中图分类号］　R733.71；R392.11　　　［文献标识码］　A

Analysis of T cell clonality by using T-cell receptor varible β gene

　　repertoire in T-cell acute lymphoblastic leukemia^·

LI Yang-qiu, CHEN Shao-hua, YANG Li-jian, QI Ming-fang

　　(Department of Hematology, Medical College, Jinan Univesity, Guangzhou 510632， China)

　　［Abstract］　 AIM: To analyze T cell clonality in patients with T cell acute lymphoblastic leukemia (T-ALL). METHODS: The complementarity determining region 3 (CDR3) size of 24 T cell antigen receptor variable β (TCR Vβ) region gene was analyzed in peripheral blood mononuclear cell (PBMC) samples from 6 T-ALL cases and 10 normal individuals by using reverse transcriptase-polymerase chain reaction (RT-PCR). The PCR products were further studied by genescan and sequencing analysis. RESULTS: Some TCR Vβ subfamily T cells display mono- or oligoclonal expansions in 3 cases of T-ALL, predominantly in Vβ2, Vβ3, Vβ6, Vβ9, Vβ21 or Vβ24, respectively. Polyclonal expansions of T cells were found in the other three cases, which could also be found in normal samples. CONCLUSION: A part of T cell acute lymphoblastic leukemia cells may arise from a clonal expansion of TCR Vβ subfamily T cell. This method may be useful for the detection of minimal residual disease in clinical study of the disease.

　　［MeSH］　 Receptors, antigen, T-cell; Genes; Leukemia

　　［CLC number］　R733.71; R392.11　　　［Document code］　A

　　［Article FD］　1000-4718(2000)07-0627-06

　　Recently, T cell receptor Vβ gene repertoire and clonality have been studied in patients with leukemia and solid tumors, by assaying the CDR3 size of T cell receptor (TCR) genes with the use of RT-PCR and genescan analysis^［1,2］. TCRs are heterodimers comprising either α/β or γ/δ chains. Like immunoglobin (Ig), by rearrangement of V(D)J-gene segments and randomly inserted nucleotides, this is termed complementarity determining region 3 (CDR3) and displays high diversity^［2］. The majority of V and J regions in human TCR have been identified. The TCR β chain gene is known to contain at least 64 functional Vβ genes subdivided into 24 Vβ families, two diversity segments (Dβ 1.1 and Dβ 2.1), and 13 joining (Jβ 1.1-1,6 and Jβ 2.1-2.7) elements. In the present study we used primers of 24 Vβ subfamilies to assay Vβ gene utilization and clonal expansion in patients with T-ALL.

　　MATERIALS AND METHODS

　　Samples　Peripheral blood mononuclear cells (PBMCs) from 6 patients with primary and untreatment T-cell acute lymphoblastic leukemia (T-ALL) were used in this study. T-cell lines Molt-4, Jurkat and K37 served as monoclonal controls, and PBMCs from 10 normal individuals served as polyclonal controls.

　　RNA extraction and cDNA synthesis　RNA was extracted according to the direction of the RNAzol Kit and reversely transcribed into the first single-strand cDNA with the use of random hexamer primer and reverse transcriptase superscript II Kit (Gibco, BRL).

　　Primers　24 Vβ and a Cβ primers used in unlabeled PCR, a fluorescent primer labeled at its 5' end with fam fluorophore (Cβ-fam) for runoff reaction and a sequencing primer labeled at its 5' end with biotin (Cβ-bio) were purchased from TIB MOLBIOL GmbH, Berlin, Germany. Nucleotide sequences of the primers are listed in Table 1^［1，3］.

　　Polymerase chain reaction (PCR)　PCR was performed as described by Puisieux et al^［1，4］. Aliquots of the cDNA (1　μL) were amplified in a 25μL reaction system with one of the 24 Vβ primer and one Cβ primer. The final reaction mixture contained 0.5 μmol/L sense primer (Vβ), 0.5 μmol/L Cβ primer, 0.1 mmol/L dNTP, 1.25 U Taq polymerase (Perkin Elmer) and 1×PCR buffer containing 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl₂ and 0.001% (w/v) gelatin. The amplification was performed on a DNA thermal cycler (Perkin Elmer). After 3 min of denaturation at 94 ℃, 40 PCR cycles were performed, each cycle consisting of reactions at 94 ℃ for 1 min, 60 ℃ for 1 min and 72 ℃, for 1 min, and a final polymerization step of 10 min at 72 ℃. The products were then stored at 4 ℃.

　　Analysis of T cell clonality

　　(1)Runoff reactions（labeled PCR products）　Aliquots of the unlabeled PCR products ( 2 μL) were separately added to a final 10 μl reaction system containing 0.1 μmol/L Cβ-fam primer, 3 mmol/L MgCl₂, 0.2 mmol/L dNTP, 0.25 U Taq polymerase and PCR buffer (Perkin Elmer). After a 3 min denaturation at 94 ℃, 35 cycles of amplification were carried out (1 min at 94 ℃, 1 min at 66 ℃ and 1 min at 72 ℃ and a final 10 min elongation at 72 ℃)^［4］.

　　(2) Genescan analysis (CDR 3 length analysis) 　The fluorescent labeled PCR products (2 μL) were heat-denatured at 94 ℃ for 4 min after addition of 2.5 μL formamide, 0.5 μL of genescan-500 tamra size standards (ABI, Perkin Elmer) and 0.5 μL of loading buffer (Dextran 50 mg/mL, EDTA 25 mmol/L, Genescan-500 Tamra Kit) and were then loaded on 6% polyacrylamide gel for size and fluorescence intensity determination by Genescan 672 analysis software on 373A DNA sequencer. Since the positions of the Vβ and Cβ primers are fixed, the length distribution observed in the PCR Vβ-Cβ products depends only on the size of the rearrangement of V-D, D-J gene segment and the randomly inserted nucleotides (V_ND_NJ). After eletrophoresis on an automated sequencer and subsequent computer analysis, the products of different size could be separated and expressed as different peaks^［4］.

　　Direct sequencing of PCR product　100 μL of the biotinylated-PCR products were purified by magnetic beads, (Dynabeads M-280 Streptavidin, Dynal A. S. Norway) and resuspended in 20 μL of distilled water. The purified products( 1 μL ) were directly sequenced by using the nonradioactive and dideoxynucleotide chain termination method, and the method described in the direction of the T-dye terminator cycle sequencing ready reaction kit with AmpliTaq DNA polymerase (ABI, Perkin Elmer ). The cycle-reaction products were dried and resuspended in 4 μL of loading buffer (4 μL recrystallized deionized formamide and 1 μL 50 mmol/L Na₂EDTA, pH 8.0/blue dextran 30 mg/mL), heat-denatured at 90 ℃ for 2 min, and loaded on a 6% polyacrylamide gel and sequenced by a model 373A DNA sequencer (ABI, Perkin Elmer).

Tab 1 　Sequence of primers used to detect TCR Vβ segments

　　RESULTS　　TCR Vβ RT-PCR analysis

　　(1) Cell lines and normal peripheral blood samples　 primary RT-PCR for the cell lines gave a positive result in only one of the Vβ primers as follows: Molt-4 with Vβ 2, Jurkat with Vβ 8 and K37 with Vβ 9. For the ten normal blood samples amplified, all Vβ subfamily-specific PCR reactions yielded positive results.

　　(2) T-ALL samples　 For the six T-ALL cases investigated, primary PCR reactions gave products from 9 to 20 of the 24 Vβ subfamilies (Table 2).

Tab 2 　The expression of TCR Vβ subfamily T cells in normal control, T-ALL and T cell lines by RT-PCR

　　Genescan analysis and sequencing　All PCR products that gave a positive band identified by 2.5% agarose gel stained with ethidium bromide, were subjected to run off reaction and genescan analysis. PCR products were separated on a polyacrylamide gel and analyzed by automatic fluorescence quantification and size-determination by using the computer program genescan 672 in the automated DNA sequencer. Because of the hypervariable character of rearranged TCRβ V-N(D)-N-J junctions, the size distribution pattern of a given PCR product should represent the characteristics of the corresponding T cell population. Products derived from homogeneous clonal cell populations, such as the cell lines and the leukemia cells, should display one or two sharp and dominant peaks of fluorescence corresponding to the PCR-amplified clonally rearranged alleles. Consequently, mRNA extracted from polyclonal T cells should yield a fluorescence spectrum of DNA bands composed of polyclonal PCR fragments of different sizes. In the present study, all Vβ family PCR products in ten normal control samples displayed multi-peak pattern (polyclonal). Mono-peak pattern (monoclonal), however, was found in all T cell line PCR products. The most of the PCR products from the 6 T-ALL cases also displayed polyclonality, whereas a part of the PCR products from 3 out of 6 samples (T-ALL case 3, 5, 6) displayed a single or dominant peak corresponding to a mono or oligoclonal CDR3 size, strongly suggesting clonal expansion of their T cells (Table 3 Fig 1). Three monoclonal PCR products, i e the Vβ 21-Cβ, Vβ 3-Cβ and Vβ 2-Cβ PCR products from case 3, 5 and 6, were further subjected to direct sequencing. The sequences are showed in Table 4.

Tab 3　 Vβ subfamilies with mono- or oligoclonal distribution of the CDR3 size

　　Note: oligo=oligoclonal; mono=monoclonal

Tab 4 　VDJ sequences of monoclonal expansions in PBL form 3 T-ALL cases

Fig 1　TCR Vβ repertoire of peripheral blood T cells obtained from case 3. 17 of the 24 Vβ subfamilies could be detected by RT-PCR. The genescan analysis shows a monoclonal T cells in Vβ 21, two oligoclonal T cells in Vβ 6 and Vβ 9, respectively, and polyclonal T cells in the other Vβ subfamilies

　　DISCUSSION

　　Skewed TCR Vβ gene segment expression in PBMCs of T-ALL　In normal humans, stable profiles of Vβ gene segments and CDR3 size were observed in T cells^［5］. In the present study, data obtained from healthy individuals indicate that all Vβ subfamilies of T cells were expressed in PBMCs. However, one significant difference between normal individuals and T-ALL cases is the change in the distribution of Vβ gene segments in the patients. Only 9 to 20 Vβ subfamily gene segments were detected by RT-PCR in blood samples from patients with T-ALL. These results suggest that skewed expression of TCR Vβ repertoire is one feature of T-ALL. It may be due to uncontrolled expansion of the malignant clonal cells, leading to suppression of the proliferation of normal T cell subfamilies and hence the inhibition of the T cell-mediated immunity.