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
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
中国病理生理杂志000714
[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。
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[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.
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[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.
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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).
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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 ℃.
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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].
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(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 (VNDNJ). After eletrophoresis on an automated sequencer and subsequent computer analysis, the products of different size could be separated and expressed as different peaks[4].
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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).
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Tab 1 Sequence of primers used to detect TCR Vβ segments Primer
Sequence
Vβ1
5'-CCGCACAACAGTTCCCTGACTTGC
Vβ2
5'-GGCCACATACGAGCAAGGCGTCGA
Vβ3
5'-CGCTTCTCCCGGATTCTGGAGTCC
Vβ4
5'-TTCCCATCAGCCGCCCAAACCTAA
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Vβ5
5'-AGCTCTGAGCTGAATGTGAACGCC
Vβ6
5'-TCTCAGGTGTGATCCAAATTCGGG
Vβ7
5'-CCTGAATGCCCCAACAGCTCTCTC
Vβ8
5'-CCATGATGCGGGGACTGGAGTTGC
Vβ9
5'-TTCCCTGGAGCTTGGTGACTCTGC
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Vβ10
5'-CCACGGAGTCAGGGGACACAGCAC
Vβ11
5'-TGCCAGGCCCTCACATACCTCTCA
Vβ12
5'-TGTCACCAGACTGGGAACCACCAC
Vβ13
5'-CACTGCGGTGTACCCAGGATATGA
Vβ14
5'-GGGCTCGGCTTAAGGCAGACCTAC
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Vβ15
5'-CAGGCACAGGCTAAATTCTCCCTG
Vβ16
5'-GCCTGCAGAACTGGAGGATTCTGG
Vβ17
5'-CTGCTGAATTTCCCAAAGAGGGCC
Vβ18
5'-TGCCCCAGAATCTCTCAGCCTCCA
Vβ19
5'-TCCTCTCACTGTGACATCGGCCCA
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Vβ20
5'-AGCTCTGAGGTGCCCCAGAATCTC
Vβ21
5'-TCCAACCTGCAAGGCTTGACGACT
Vβ22
5'-AAGTGATCTTGCGCTGTGTCCCCA
Vβ23
5'-GCAGGGTCCAGGTCAGGACCCCCA
Vβ24
5'-CCCAGTTTGGAAAGCCAGTGACCC
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Cβ
5'-CGGGCTGCTCCTTGAGGGGCTGCG
Cβ-fam
5'-Fam-CACAGCGACCTCGGGTGGG
Cβ-bio
5'-Bio-CACAGCGACCTCGGGTGGGAA
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.
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(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 Primer
Normal
case
Case 1
Case 2
Case 3
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Case 4
Case 5
Case 6
Molt-4
Jurkat
K37
Vβ1/Cβ
+
+
+
+
+
+
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+
Vβ2/Cβ
+
+
+
+
+
+
+
+
Vβ3/Cβ
+
+
+
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+
+
+
+
Vβ4/Cβ
+
+
Vβ5/Cβ
+
+
+
+
+
+
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Vβ6/Cβ
+
+
+
+
Vβ7/Cβ
+
+
+
Vβ8/Cβ
+
+
+
+
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Vβ9/Cβ
+
+
+
+
+
Vβ10/Cβ
+
+
Vβ11/Cβ
+
+
+
+
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Vβ12/Cβ
+
+
Vβ13/Cβ
+
+
+
+
+
+
+
Vβ14/Cβ
+
+
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+
+
+
+
Vβ15/Cβ
+
+
+
+
+
+
+
Vβ16/Cβ
+
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+
+
+
+
+
Vβ17/Cβ
+
+
+
+
+
Vβ18/Cβ
+
+
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+
+
Vβ19/Cβ
+
+
+
+
+
Vβ20/Cβ
+
Vβ21/Cβ
+
+
+
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+
+
+
Vβ22/Cβ
+
+
+
+
+
+
+
Vβ23/Cβ
+
+
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+
+
+
Vβ24/Cβ
+
+
+
+
+
+
+
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 Primer
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Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Molt-4
Jurkat
K37
Vβ2/Cβ
mono
mono
Vβ3/Cβ
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mono
Vβ6/Cβ
oligo
Vβ8/Cβ
mono
Vβ9/Cβ
oligo
mono
Vβ21/Cβ
mono
Vβ24/Cβ
oligo
Note: oligo=oligoclonal; mono=monoclonalTab 4 VDJ sequences of monoclonal expansions in PBL form 3 T-ALL cases Segment
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Vβ
Sequence
Case 6: GAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCT G AAGACAGCAGCTTCTACATCTGCAG
Case 5:............................................................CCACCACCAACCAGCATCATCTATGTACCTCTGTGCCACCAGCTC
Case 3: ....................................................................................CGGCCGTGTATCTCTGTGCCAGC
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Segment
N
Dβ
N
Jβ
Sequence
TGCTACAGGAT
GGGAGGG
G
ACGACGACTACTTCTTCGGGGCCG GGCA CCAGGCTCCGGCTCATGGTCAACVβ2NDβ2.1NJβ2.7
AT
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ACAGGGG
TTGAG
ACAGGAAAAACTCTTTTTTGGCACTGGAACCCAGCTCTCTGTCTTG Vβ3NDβ1.1NJβ1.4
CTCAC
GGAC
GGAG
TACAATGAGCAGTTCTTCGGGCCAGG GACACGGCTCACCGTGCTA Vβ21NDβ2.1NJβ2.1
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
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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.
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Clonal expansion in T-ALL T cell acute lymphoblastic leukemia is generally considered to be a malignant clonal disorder arising from an uncontrolled expansion of committed lymphoid precursors. In the present study, a highly sensitive method, which is considered at present now as the best means for the identification of T cell clonality internationally, was used to analyze T cell clonality in PBMCs from 6 T-ALL patients. However, clonal expansion of TCR Vβ subfamilies was found only in 3 samples. Genescan analysis of the 3 T-ALL patients revealed that monoclonal Vβ subfamily T cells were Vβ 2(Case6), Vβ 3(Case5) and Vβ 21(Case3) respectively. These results provide strong evidence for the presence of neoplastic T cell monoclonality, which was confirmed by subsequent direct sequencing of the PCR products. Besides two oligoclonal T cells subfamilies, Vβ 6 and Vβ 9, were found in case 3, while one oligoclonal T cell subfamily, Vβ 24 was present in Case6. Recent studies have showed that oligoclonal expansion of T cells could be found in non-T cell leukemias, including acute non-lymphocytic leukemia (ANLL) and B cell-chronic lymphocytic leukemia (B-CLL)[4，6]. The appearance of such oligoclonal T cells may reflect the hosts T cell response to leukemia associated antigen. These stimulated normal T cell populations are considered to have specific capability of coping with leukemia cells. The existence of one or two Vβ subfamily oligoclonal T cells in two T-ALL cases led us to postulate that they may be specific anti-T-ALL T cells or leukemia-reactive T cells[7]. The results also showed that clonal expansion of T cells of the Vβ subfamilies was different among different patients, which may be attributed to individuality of the immune response in different persons. Clonal expansion of T cells could not be detected by the CDR3 size analysis of TCR Vβrepertoire in the remaining 3 cases of T-ALL. Instead, only polyclonal TCR Vβ subfamily T cells were found in 3 patients. It is possible that clonal T cells of other TCR subfamily, for example, TCRγ or δ may be present. The technique used in the present study, together with its corresponding specific primers, may also be applied to the analysis of the CDR3 size of TCR α, γ or δ.
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A molecular biologic marker for detecting clonal evolution and minimal residual disease The technique used in the present study has several advantages over conventional Southern blot analysis, including speed, safety, sensitivity, and no radioactivity. It may also prove valuable in its clinical application. For example, the appearance of malignant clonal cells in leukemia patients undergoing relapse may be readily detected at the early stage by the specific primers for the Vβ gene. It may be used as an important means for the detection of minimal residual disease (MRD) and in confirmation of clonal evolution of T-ALL cells during relapse, which is very important for the chemotherapy design[7].
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In conclusion, using such an approach to analyze the TCR Vβ repertoire, we may obtain a global picture of the distribution, clonality and CDR3 combinations of TCR Vβ repertoire in the PBMCs from patients with T-ALL. The technique is very useful to the understanding of the skew expression, clonal expansion of leukemia or anti-leukemia cells in T-ALL, as well as to the detection of minimal residual disease in clinical investigation.
[Foundation item] This work was supported by the grants from the national natural science foundation of China (No. 39870358); the natural science foundation of Guangdong province (No. 980705); the major research project of Guangzhou science technology committee(No. 98-z-039-01)
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[REFERENCES]
[1] Puiseux I, Even J, Pannetier C, et al. Oligoclonality of tumor-infiltrating lymphocytes from human melanomas[J]. J Immunol, 1994, 153:2807～2818.
[2] Sensi M, Parmiani G. Analysis of TCR usage in human tumors: a new tool for assessing tumor-specific immune response[J]. Immunol Today, 1995, 16:588～595.
[3] Liu X, Chesnokova V, Forman SJ, et al. Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions[J]. Blood, 1996,87:3032～3044.
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[4] 李扬秋, 汪明春，Siegert W,等. 利用基因扫描分析TCR Vβ 亚家族 CDR3长度的方法检测AML的T细胞克隆性[J]. 肿瘤, 1997, 17:435～438.
[5] Liu D, Callahan JP, Dau PC. Intrafamily fragment analysis of the T cell receptor β chain CDR3 region[J]. J Immunol Methods, 1995, 187:139～150.
[6] Farace F, Orlanducci F, Dietrich PT, et al. T cell repertoire in patients with B chronic lymphocytic leukemia[J]. J Immunol, 1994, 153:4281～4291.
[7] Prevost-Blondel A, Ostankovitch M, Melle J, et al. CDR3 size analysis of T cell receptor V beta transcripts: follow-up study in a patient with T cell acute lymphoblastic leukemia[J]. Leukemia, 1995, 9:1711～1717.
[Received date]1999-05-06
[Accepted date]1999-07-22, http://www.100md.com
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
中国病理生理杂志000714
[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。
, http://www.100md.com
[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.
, 百拇医药
[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.
, http://www.100md.com
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).
, 百拇医药
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 ℃.
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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].
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(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 (VNDNJ). After eletrophoresis on an automated sequencer and subsequent computer analysis, the products of different size could be separated and expressed as different peaks[4].
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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).
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Tab 1 Sequence of primers used to detect TCR Vβ segments Primer
Sequence
Vβ1
5'-CCGCACAACAGTTCCCTGACTTGC
Vβ2
5'-GGCCACATACGAGCAAGGCGTCGA
Vβ3
5'-CGCTTCTCCCGGATTCTGGAGTCC
Vβ4
5'-TTCCCATCAGCCGCCCAAACCTAA
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Vβ5
5'-AGCTCTGAGCTGAATGTGAACGCC
Vβ6
5'-TCTCAGGTGTGATCCAAATTCGGG
Vβ7
5'-CCTGAATGCCCCAACAGCTCTCTC
Vβ8
5'-CCATGATGCGGGGACTGGAGTTGC
Vβ9
5'-TTCCCTGGAGCTTGGTGACTCTGC
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Vβ10
5'-CCACGGAGTCAGGGGACACAGCAC
Vβ11
5'-TGCCAGGCCCTCACATACCTCTCA
Vβ12
5'-TGTCACCAGACTGGGAACCACCAC
Vβ13
5'-CACTGCGGTGTACCCAGGATATGA
Vβ14
5'-GGGCTCGGCTTAAGGCAGACCTAC
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Vβ15
5'-CAGGCACAGGCTAAATTCTCCCTG
Vβ16
5'-GCCTGCAGAACTGGAGGATTCTGG
Vβ17
5'-CTGCTGAATTTCCCAAAGAGGGCC
Vβ18
5'-TGCCCCAGAATCTCTCAGCCTCCA
Vβ19
5'-TCCTCTCACTGTGACATCGGCCCA
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Vβ20
5'-AGCTCTGAGGTGCCCCAGAATCTC
Vβ21
5'-TCCAACCTGCAAGGCTTGACGACT
Vβ22
5'-AAGTGATCTTGCGCTGTGTCCCCA
Vβ23
5'-GCAGGGTCCAGGTCAGGACCCCCA
Vβ24
5'-CCCAGTTTGGAAAGCCAGTGACCC
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Cβ
5'-CGGGCTGCTCCTTGAGGGGCTGCG
Cβ-fam
5'-Fam-CACAGCGACCTCGGGTGGG
Cβ-bio
5'-Bio-CACAGCGACCTCGGGTGGGAA
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.
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(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 Primer
Normal
case
Case 1
Case 2
Case 3
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Case 4
Case 5
Case 6
Molt-4
Jurkat
K37
Vβ1/Cβ
+
+
+
+
+
+
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+
Vβ2/Cβ
+
+
+
+
+
+
+
+
Vβ3/Cβ
+
+
+
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+
+
+
+
Vβ4/Cβ
+
+
Vβ5/Cβ
+
+
+
+
+
+
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Vβ6/Cβ
+
+
+
+
Vβ7/Cβ
+
+
+
Vβ8/Cβ
+
+
+
+
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Vβ9/Cβ
+
+
+
+
+
Vβ10/Cβ
+
+
Vβ11/Cβ
+
+
+
+
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Vβ12/Cβ
+
+
Vβ13/Cβ
+
+
+
+
+
+
+
Vβ14/Cβ
+
+
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+
+
+
+
Vβ15/Cβ
+
+
+
+
+
+
+
Vβ16/Cβ
+
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+
+
+
+
+
Vβ17/Cβ
+
+
+
+
+
Vβ18/Cβ
+
+
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+
+
Vβ19/Cβ
+
+
+
+
+
Vβ20/Cβ
+
Vβ21/Cβ
+
+
+
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+
+
+
Vβ22/Cβ
+
+
+
+
+
+
+
Vβ23/Cβ
+
+
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+
+
+
Vβ24/Cβ
+
+
+
+
+
+
+
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 Primer
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Case 1
Case 2
Case 3
Case 4
Case 5
Case 6
Molt-4
Jurkat
K37
Vβ2/Cβ
mono
mono
Vβ3/Cβ
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mono
Vβ6/Cβ
oligo
Vβ8/Cβ
mono
Vβ9/Cβ
oligo
mono
Vβ21/Cβ
mono
Vβ24/Cβ
oligo
Note: oligo=oligoclonal; mono=monoclonalTab 4 VDJ sequences of monoclonal expansions in PBL form 3 T-ALL cases Segment
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Vβ
Sequence
Case 6: GAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCT G AAGACAGCAGCTTCTACATCTGCAG
Case 5:............................................................CCACCACCAACCAGCATCATCTATGTACCTCTGTGCCACCAGCTC
Case 3: ....................................................................................CGGCCGTGTATCTCTGTGCCAGC
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Segment
N
Dβ
N
Jβ
Sequence
TGCTACAGGAT
GGGAGGG
G
ACGACGACTACTTCTTCGGGGCCG GGCA CCAGGCTCCGGCTCATGGTCAACVβ2NDβ2.1NJβ2.7
AT
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ACAGGGG
TTGAG
ACAGGAAAAACTCTTTTTTGGCACTGGAACCCAGCTCTCTGTCTTG Vβ3NDβ1.1NJβ1.4
CTCAC
GGAC
GGAG
TACAATGAGCAGTTCTTCGGGCCAGG GACACGGCTCACCGTGCTA Vβ21NDβ2.1NJβ2.1
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
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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.
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Clonal expansion in T-ALL T cell acute lymphoblastic leukemia is generally considered to be a malignant clonal disorder arising from an uncontrolled expansion of committed lymphoid precursors. In the present study, a highly sensitive method, which is considered at present now as the best means for the identification of T cell clonality internationally, was used to analyze T cell clonality in PBMCs from 6 T-ALL patients. However, clonal expansion of TCR Vβ subfamilies was found only in 3 samples. Genescan analysis of the 3 T-ALL patients revealed that monoclonal Vβ subfamily T cells were Vβ 2(Case6), Vβ 3(Case5) and Vβ 21(Case3) respectively. These results provide strong evidence for the presence of neoplastic T cell monoclonality, which was confirmed by subsequent direct sequencing of the PCR products. Besides two oligoclonal T cells subfamilies, Vβ 6 and Vβ 9, were found in case 3, while one oligoclonal T cell subfamily, Vβ 24 was present in Case6. Recent studies have showed that oligoclonal expansion of T cells could be found in non-T cell leukemias, including acute non-lymphocytic leukemia (ANLL) and B cell-chronic lymphocytic leukemia (B-CLL)[4，6]. The appearance of such oligoclonal T cells may reflect the hosts T cell response to leukemia associated antigen. These stimulated normal T cell populations are considered to have specific capability of coping with leukemia cells. The existence of one or two Vβ subfamily oligoclonal T cells in two T-ALL cases led us to postulate that they may be specific anti-T-ALL T cells or leukemia-reactive T cells[7]. The results also showed that clonal expansion of T cells of the Vβ subfamilies was different among different patients, which may be attributed to individuality of the immune response in different persons. Clonal expansion of T cells could not be detected by the CDR3 size analysis of TCR Vβrepertoire in the remaining 3 cases of T-ALL. Instead, only polyclonal TCR Vβ subfamily T cells were found in 3 patients. It is possible that clonal T cells of other TCR subfamily, for example, TCRγ or δ may be present. The technique used in the present study, together with its corresponding specific primers, may also be applied to the analysis of the CDR3 size of TCR α, γ or δ.
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A molecular biologic marker for detecting clonal evolution and minimal residual disease The technique used in the present study has several advantages over conventional Southern blot analysis, including speed, safety, sensitivity, and no radioactivity. It may also prove valuable in its clinical application. For example, the appearance of malignant clonal cells in leukemia patients undergoing relapse may be readily detected at the early stage by the specific primers for the Vβ gene. It may be used as an important means for the detection of minimal residual disease (MRD) and in confirmation of clonal evolution of T-ALL cells during relapse, which is very important for the chemotherapy design[7].
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In conclusion, using such an approach to analyze the TCR Vβ repertoire, we may obtain a global picture of the distribution, clonality and CDR3 combinations of TCR Vβ repertoire in the PBMCs from patients with T-ALL. The technique is very useful to the understanding of the skew expression, clonal expansion of leukemia or anti-leukemia cells in T-ALL, as well as to the detection of minimal residual disease in clinical investigation.
[Foundation item] This work was supported by the grants from the national natural science foundation of China (No. 39870358); the natural science foundation of Guangdong province (No. 980705); the major research project of Guangzhou science technology committee(No. 98-z-039-01)
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[REFERENCES]
[1] Puiseux I, Even J, Pannetier C, et al. Oligoclonality of tumor-infiltrating lymphocytes from human melanomas[J]. J Immunol, 1994, 153:2807～2818.
[2] Sensi M, Parmiani G. Analysis of TCR usage in human tumors: a new tool for assessing tumor-specific immune response[J]. Immunol Today, 1995, 16:588～595.
[3] Liu X, Chesnokova V, Forman SJ, et al. Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions[J]. Blood, 1996,87:3032～3044.
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[4] 李扬秋, 汪明春，Siegert W,等. 利用基因扫描分析TCR Vβ 亚家族 CDR3长度的方法检测AML的T细胞克隆性[J]. 肿瘤, 1997, 17:435～438.
[5] Liu D, Callahan JP, Dau PC. Intrafamily fragment analysis of the T cell receptor β chain CDR3 region[J]. J Immunol Methods, 1995, 187:139～150.
[6] Farace F, Orlanducci F, Dietrich PT, et al. T cell repertoire in patients with B chronic lymphocytic leukemia[J]. J Immunol, 1994, 153:4281～4291.
[7] Prevost-Blondel A, Ostankovitch M, Melle J, et al. CDR3 size analysis of T cell receptor V beta transcripts: follow-up study in a patient with T cell acute lymphoblastic leukemia[J]. Leukemia, 1995, 9:1711～1717.
[Received date]1999-05-06
[Accepted date]1999-07-22, http://www.100md.com