Clonal Expansion and Cytotoxicity of TCRVβ Subfamily T Cells Induced by CML and K562 Cells

Samples

Peripheral blood mononuclear cells (PBMCs) from one patient with primary and untreated chronic myelogenous leukemia who expressed b3a2 BCR-ABL fusion gene, determined by RT-PCR, were used in this study. The diagnosis of CML was based on the morphology and genetics. Three cases of cord blood (Q1–Q3) were collected from the umbilical vein of neonates after normal full-term deliveries according to informed consent guidelines (maternal consent was obtained in all cases). No HBV, HCV or CMV infections were found in peripheral blood from the mothers before delivery. Peripheral blood was obtained from a healthy adult blood donor. K562 and Raji cell lines were provided from the Hematology Lab of Nan Fang Hospital or Wuhan University Cells Bank, respectively.

Mononuclear cell isolation

Cord blood and adult peripheral blood mononuclear cells(CBMC or PBMC) were isolated by density gradient centrifugation(Ficoll-Hypaque density 1.077 g/ml). CD3^- cells(CML cells) were negatively separated by using a Mini magnetic bead sorter according to the manufacturer’s instructions (MACs, Miltenyi Biotec, Germany).

Mixed Lymphocyte-leukemia cell cultures

CBMC or PBMC at a concentration of 1×10⁶/ml were divided into three groups respectively. Each group was stimulated respectively with K562 cells or CML cells, or without any additional cells, at a responder-stimulator ratio of 10:1. CML cells and K562 cells used as stimulators were treated with 50mg/ml mitomycin C (MMC) for 1 hour and washed 4 times by RPMI–1640 containing 15% human male AB serum. Cell cultures were incubated at 37°C in a humidified atmosphere with 5% CO₂, in a total volume of 2ml/well of RPMI-1640 supplemented with 100 IU penicillin, 100μg/ml streptomycin, 10% human serum albumin, 1μg/ml anti–CD3 McAb, 500 U/ml IL–2, 50 μmol/L 2–ME, 2 mmol/L L-glutamine and 1μg/ml anti–CD28 McAb. On day 3–5 of incubation, half of the medium was exchanged for culture medium supplemented with 100 IU penicillin, 100μg/ml streptomycin, 2% human serum albumin, 500 U/ml IL-2, and 2 mmol/L L-glutamine. The second and third stimulations were performed at 5-day intervals with MMC-treated K562 cells or CML cells respectively and half medium exchanged simultaneously.

Target cell culture

Raji and K562 cell line, CML cells selected by mini MACS were cultured at 1 x10⁶/ml with RPMI–1640 media (Gibco, BRL) containing 10% human male AB serum, 100 IU penicillin and 100 μg/ml streptomycin.

T cell phenotype analysis

T cell phenotype was analyzed by indirect immunophenotyping fluorescein dyeing using anti-CD3, CD4 and CD8 antibody and fluorescence microscopy.

T-cell cytotoxicity assay CTL

T–cell cytotoxicity was measured in vitro using the lactate dehydrogenase(LDH) release assay. Target cells were cocultured with effector T cells at effector-target cell ratio of 30:1 for 4 hours in 96-well round-bottom plates. Spontaneous release of effector and target cells was controlled by separate incubation of the respective populations. Maximal LDH enzyme release was measured after lysis of the target cells with 0.5% Triton X-100 (Sigma). Cell-free 100 μl supernatants were incubated in a separate 96-well plate with LDH 100 μl substrate for 30 min. at room temperature and measured by a microplate reader (Elx-800) at 490 nm with 650 nm reference. The percentage of cytotoxicity was calculated according to the following formula: n=([E-St-Se]/[M–St])×100 (%), (E: LDH release by effector–target co-culture, St: the spontaneous release by target cells, Se: the spontaneous release by effector cells, M: the maximal release by target cells).

RNA extraction and cDNA synthesis

RNA was extracted according to the direction of the Trizol 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).

Polymerase chain reaction (PCR)

Nucleotide sequences of the 24 Vβ primers and a Cβ primer were used in unlabeled PCR. A fluorescent primer labeled at its 5’ end with fam fluorophore (Cβ-fam) for runoff reaction was purchased from TIB MOLBIOL GmbH, Berlin, Germany^[5–6]. PCR was performed as described by Puisieux I et al ^[5–6]. Aliquots of the cDNA (1μL) were amplified in a 25μL reaction system with one 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, 50mmol/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°C, 40 PCR cycles were performed, each cycle consisting of reactions at 94°C for 1 min, 60°C for 1 min and 72°C, for 1 min, and a final polymerization step of 10 min at 72°C. The products were then stored at 4t.

Analysis of T cell clonality

Runoff reactions (labeled PCR products) Aliquots of the unlabeled PCR product (2μL) 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 94t, 35 cycles of amplification were carried out (1 min at 94°C, 1 min at 66°C and 1 min at 72°C and a final 10 min elongation at 72°C^[5–6].

Genescan analysis(CDR 3 length analysis) The fluorescent labeled PCR products (2 μL) were heat–denatured at 94°C 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 50mg/ml, EDTA 25mmol/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 a 377A 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^[5–6].