受体变异β基因谱系分析T细胞急性淋巴细胞白血病病.doc

利用T细胞受体变异基因谱系分析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(TCR V)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 V亚家族T细胞呈单克隆或寡克隆性增殖，主要为V2、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 V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AAnalysis of T cell clonality by using T-cell receptor varible generepertoire in T-cell acute lymphoblastic leukemiaLI 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 (TCR V) 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 V subfamily T cells display mono- or oligoclonal expansions in 3 cases of T-ALL, predominantly in V2, V3, V6, V9, V21 or V24, 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 V 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; LeukemiaCLC numberR733.71; R392.11Document codeAArticle FD1000-4718(2000)07-0627-06Recently, T cell receptor V 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 analysis1,2. TCRs are heterodimers comprising either / or / 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 diversity2. The majority of V and J regions in human TCR have been identified. The TCR chain gene is known to contain at least 64 functional V genes subdivided into 24 V families, two diversity segments (D 1.1 and D 2.1), and 13 joining (J 1.1-1,6 and J 2.1-2.7) elements. In the present study we used primers of 24 V subfamilies to assay V gene utilization and clonal expansion in patients with T-ALL.MATERIALS AND METHODSSamplesPeripheral 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 synthesisRNA 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).Primers24 V and a C primers used in unlabeled PCR, a fluorescent primer labeled at its 5 end with fam fluorophore (C-fam) for runoff reaction and a sequencing primer labeled at its 5 end with biotin (C-bio) were purchased from TIB MOLBIOL GmbH, Berlin, Germany. Nucleotide sequences of the primers are listed in Table 11，3.Polymerase chain reaction (PCR)PCR was performed as described by Puisieux et al1，4. Aliquots of the cDNA (1L) were amplified in a 25L reaction system with one of the 24 V primer and one C primer. The final reaction mixture contained 0.5 mol/L sense primer (V), 0.5 mol/L C primer, 0.1 mmol/L dNTP, 1.25 U Taq polymerase (Perkin Elmer) and 1PCR 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 L) were separately added to a final 10 l reaction system containing 0.1 mol/L C-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 L) were heat-denatured at 94 for 4 min after addition of 2.5 L formamide, 0.5 L of genescan-500 tamra size standards (ABI, Perkin Elmer) and 0.5 L 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 V and C primers are fixed, the length distribution observed in the PCR V-C 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 peaks4.Direct sequencing of PCR product100 L of the biotinylated-PCR products were purified by magnetic beads, (Dynabeads M-280 Streptavidin, Dynal A. S. Norway) and resuspended in 20 L of distilled water. The purified products( 1 L ) 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 L of loading buffer (4 L recrystallized deionized formamide and 1 L 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 V segmentsPrimerSequence V15-CCGCACAACAGTTCCCTGACTTGCV25-GGCCACATACGAGCAAGGCGTCGAV35-CGCTTCTCCCGGATTCTGGAGTCCV45-TTCCCATCAGCCGCCCAAACCTAAV55-AGCTCTGAGCTGAATGTGAACGCCV65-TCTCAGGTGTGATCCAAATTCGGGV75-CCTGAATGCCCCAACAGCTCTCTCV85-CCATGATGCGGGGACTGGAGTTGCV95-TTCCCTGGAGCTTGGTGACTCTGCV105-CCACGGAGTCAGGGGACACAGCACV115-TGCCAGGCCCTCACATACCTCTCAV125-TGTCACCAGACTGGGAACCACCACV135-CACTGCGGTGTACCCAGGATATGAV145-GGGCTCGGCTTAAGGCAGACCTACV155-CAGGCACAGGCTAAATTCTCCCTGV165-GCCTGCAGAACTGGAGGATTCTGGV175-CTGCTGAATTTCCCAAAGAGGGCCV185-TGCCCCAGAATCTCTCAGCCTCCAV195-TCCTCTCACTGTGACATCGGCCCAV205-AGCTCTGAGGTGCCCCAGAATCTCV215-TCCAACCTGCAAGGCTTGACGACTV225-AAGTGATCTTGCGCTGTGTCCCCAV235-GCAGGGTCCAGGTCAGGACCCCCAV245-CCCAGTTTGGAAAGCCAGTGACCCC5-CGGGCTGCTCCTTGAGGGGCTGCGC-fam5-Fam-CACAGCGACCTCGGGTGGGC-bio5-Bio-CACAGCGACCTCGGGTGGGAARESULTSTCR V 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 V primers as follows: Molt-4 with V 2, Jurkat with V 8 and K37 with V 9. For the ten normal blood samples amplified, all V 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 V subfamilies (Table 2).Tab 2 The expression of TCR V subfamily T cells in normal control, T-ALL and T cell lines by RT-PCRPrimerNormal caseCase 1Case 2Case 3Case 4Case 5Case 6Molt-4JurkatK37V1/C+V2/C+V3/C+V4/C+V5/C+V6/C+V7/C+V8/C+V9/C+V10/C+V11/C+V12/C+V13/C+V14/C+V15/C+V16/C+V17/C+V18/C+V19/C+V20/C+V21/C+V22/C+V23/C+V24/C+Genescan analysis and sequencingAll 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 TCR 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 V 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 V 21-C, V 3-C and V 2-C PCR products from case 3, 5 and 6, were further subjected to direct sequencing. The sequences are showed in Table 4.Tab 3 V subfamilies with mono- or oligoclonal distribution of the CDR3 sizePrimerCase 1Case 2Case 3Case 4Case 5Case 6Molt-4JurkatK37V2/CmonomonoV3/CmonoV6/ColigoV8/CmonoV9/ColigomonoV21/CmonoV24/ColigoNote: oligo=oligoclonal; mono=monoclonal Tab 4 VDJ sequences of monoclonal expansions in PBL form 3 T-ALL casesSegmentVSequenceCase 6: GAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCT G AAGACAGCAGCTTCTACATCTGCAGCase 5:.CCACCACCAACCAGCATCATCTATGTACCTCTGTGCCACCAGCTCCase3: .CGGCCGTGTATCTCTGTGCCAGCSegmentN DN JSequenceTGCTACAGGATGGGAGGG G ACGACGACTACTTCTTCGGGGCCG GGCA CCAGGCTCCGGCTCATGGTCAAC V2ND2.1NJ2.7ATACAGGGGTTGAG ACAGGAAAAACTCTTTTTTGGCACTGGAACCCAGCTCTCTGTCTTG V3ND1.1NJ1.4CTCAC GGAC GGAG TACAATGAGCAGTTCTTCGGGCCAGG GACACGGCTCACCGTGCTA V21ND2.1NJ2.1Fig 1TCR V repertoire of peripheral blood T cells obtained from case 3. 17 of the 24 V subfamilies could be detected by RT-PCR. The genescan analysis shows a monoclonal T cells in V 21, two oligoclonal T cells in V 6 and V 9, respectively, and polyclonal T cells in the other V subfamiliesDISCUSSIONSkewed TCR V gene segment expression in PBMCs of T-ALLIn normal humans, stable profiles of V gene segments and CDR3 size were observed in T cells5. In the present study, data obtained from healthy individuals indicate that all V 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 V gene segments in the patients. Only 9 to 20 V subfamily gene segments were detected by RT-PCR in blood samples from patients with T-ALL. These results suggest that skewed expression of TCR V 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.Clonal expansion in T-ALLT 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 V subfamilies was found only in 3 samples. Genescan analysis of the 3 T-ALL patients revealed that monoclonal V subfamily T cells were V 2(Case6), V 3(Case5) and V 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, V 6 and V 9, were found in case 3, while one oligoclonal T cell subfamily, V 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 V 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 cells7. The results also showed that clonal expansion of T cells of the V 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 Vrepertoire in the remaining 3 cases of T-ALL. Instead, only polyclonal TCR V subfamily T cells were found in 3 patients. It is possible that clonal T cells of other TCR subfamily, for example, TCR or 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 , or .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 V 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 design7.In conclusion, using such an approach to analyze the TCR V repertoire, we may obtain a global picture of the distribution, clonality and CDR3 combinations of TCR V repertoire in the PBMCs