Sensitivity and Resistance to Therapy

In vitro IL-12 treatment of peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes: increase in cytotoxicity and reduction in WT1 gene expression

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Abstract

Interleukin-12 (IL-12) has potent antitumor activities. We examined whether IL-12 enhanced the cytotoxicity of peripheral blood mononuclear cells (PBMNC) and decreased leukemia cells in 30 patients with leukemia or myelodysplastic syndromes (MDS): 12 acute myeloid leukemia (AML) (five in complete remission (CR) and seven in non-CR); six chronic myeloid leukemia (CML); and 12 MDS (three refractory anemia (RA), eight RA with excess of blasts and one chronic myelomonocytic leukemia). PBMNC from patients and five healthy volunteers were cultured at 5 × 105/ml parallel with or without 100 units/ml of IL-12 for 3 days. Cytotoxicity of PBMNC against K562 cells was assessed by flow cytometry. To quantify the amount of leukemia cells, WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR), since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia or MDS. The cytotoxicity of non-IL-12-treated PBMNC of 30 patients was 13.4 ± 9.3% at the effector to target (E:T) ratio of 20:1, and significantly lower than that of normal subjects (25.7 ± 8.4%). The cytotoxicity increased to 30.6 ± 17.9% in the IL-12-treated PBMNC. WT1 mRNA in PBMNC of five healthy volunteers was less than 103 copies/μg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 104.8 to 104.2copies/μg of total RNA in six CML patients, from 105.4 to 104.8copies/μg in 12 MDS patients and from 105.0 to 104.2 copies/μg in five AML patients in CR, but not reduced in five of seven AML in non-CR. These results showed that IL-12 significantly enhanced PBMNC cytotoxicity and decreased the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 might be of considerable benefit in the elimination of MRD in patients with hematological malignancies. Leukemia (2000) 14, 1634–1641.

Introduction

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by monocytes, B lymphocytes and other antigen-presenting cells under physiological conditions.12 It was initially given the names of natural killer (NK) cell stimulatory factor or cytotoxic lymphocyte maturation factor based on its stimulatory effects on these cytolytic lymphocyte populations. In vitro studies have demonstrated that IL-12 can enhance the non-MHC-restricted cytolytic activity of NK cells3 and facilitate specific allogeneic human cytolytic T lymphocyte responses against fresh leukemia cells and cell lines.456 IL-12 can potently induce IFN-γ,78 and up-regulate the expression of cytolytic components including perforin, serine esterase and TIA-1 in peripheral blood lymphocytes.9 IL-12 can also stimulate continuous proliferation of both T lymphocytes and NK cells.10

IL-12 has been shown to have potent antitumor activities in murine tumor models, including B16F10 melanoma, renal cell adenocarcinoma, M5076 reticulum cell sarcoma, RAW117-H10 lymphoma and fibrosacoma.11121314 In the RAW117-H10 lymphoma model, IL-12 showed a significant antitumor effect against metastatic lymphoma in the liver of lymphoma- bearing mice that had been treated with high-dose chemotherapy followed by stem cell transplantation, a situation which mimics clinical minimal residual disease (MRD).13 Subcutaneous administration of IL-12 also showed promising antitumor effects in patients with cutaneous T cell lymphoma15 and metastatic renal cell carcinoma.161718 These data suggest that IL-12 may be useful in the therapy of patients with hematological malignancies.

The Wilms’ tumor gene, WT1, is a tumor suppressor gene located at chromosome 11p13. This gene is involved in growth control and differentiation of various types of cells. The expression of WT1 gene is reportedly restricted to selective tissues, including Sertoli cells, decidua cells of the uterus, and mesothelial cells. Recent studies, however, showed that almost all human leukemia samples examined expressed WT1 mNA, regardless of the disease subtype.192021 WT1 gene expression in leukemia cells is approximately 105 times higher than in normal peripheral blood (PB) cells19 and is inversely correlated with the prognosis of acute leukemia and MDS.222324 Suppression of WT1 gene expression by WT1 antisense oligonucleotide inhibited proliferation of leukemia cells,25 implicating the involvement of WT1 in leukemogenesis. These findings suggested that WT1 mRNA could be used as a new marker of MRD in patients with leukemia or MDS. Meanwhile, it was also found that the detection sensitivity of MRD in PB is more than 100 times higher than in bone marrow (BM) because of the presence of WT-positive CD34+ cells in BM.19

The present study is thus designed to examine the cytotoxic effect of IL-12-treated PBMNC on leukemia cells in patients with leukemia or MDS to see whether IL-12 could be used in the elimination of MRD in these patients.

Materials and methods

Reagents

A recombinant human IL-12 (Hoffmann-La Roche, New Jersey, USA) with a specific activity of 2.7 × 108 units/mg, was dissolved in RPMI 1640 (Gibco, Grand Island, NY, USA) supplemented with 1% penicillin and streptomycin, 1 mM L-glutamine and 10% heat-inactivated fetal bovine serum (Gibco) (complete medium) to yield a 100 units/μl working solution. The lipophilic carbocyanine membrane dye 3,3′-dioctadecyloxacarbocyanine perchlorate (Dio; Sigma, St Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO; Sigma) to yield a 3 mM Dio working solution, and propidium iodide (PI; Sigma) in complete medium to yield a 0.1 mg/ml working solution.

Patients and healthy volunteers

Heparinized peripheral blood was obtained after informed consent from five healthy volunteers, 12 patients with MDS (three refractory anemia (RA), eight RA with excess of blasts (RAEB) and one chronic myelomonocytic leukemia (CMML)); 12 with acute myeloid leukemia (AML) (two M1, five M2, three M3, one M4 and one M7 according to the French– American–British classification, five patients in complete remission (CR), seven in non-CR); and six with chronic myeloid leukemia in chronic phase (CML). Patients’ profiles are summarized in Table 1. Their peripheral blood contained 0–94% of blasts. PBMNC were isolated by density-gradient centrifugation with Ficoll–Paque (Pharmacia Biotech, Uppsala, Sweden) and cultured at a density of 5 × 105 cells/ml of complete medium parallel with or without 100 units of IL-12 at 37°C in a humidified atmosphere containing 5% CO2 for 3 days. Then, the following procedures were performed.

Table 1  Patients’ characteristics

Competitive RT-PCR analysis of WT1 mRNA in K562 cells and PBMNC

A human erythroleukemia cell line, K562, a well-known NK-sensitive and constitutively WT1 gene-expressing cell line,1926 was obtained from the Japanese Cancer Research Bank and used in the present study. Extraction of total RNA was performed from about 5–10 × 106 cells of K562 or PBMNC using TRI REAGENT (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. Extracted total RNA was dissolved in diethylpyrocarbonate-treated water (DEPC-water) and quantitated, and then competitive reverse transcription polymerase chain reaction (RT-PCR) was performed. Briefly, equal amounts (1 μg each) of total RNA from each sample were dissolved in DEPC-water (5 μl each) and mixed with 10 μl of 107, 106, 105, 104, 103 and 0 copies of competitor RNA (SRL, Tokyo, Japan), respectively. The mixtures were heated at 68°C for 10 min and then mixed with 15 μl of RT buffer (50 mM Tris-HCl (pH 8.3); 70 mM KCl; 3 mM MgCl2; 10 mM dithiothreitol) containing 400 units of Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Gaitherburg, MD, USA), 200 mM each of deoxyribonucleotide (dNTP; Roche Molecular Systems, Branchburg, NJ, USA), 750 ng of random-hexamer (Takara, Shiga, Japan) and 40 units of RNase inhibitor (Toyobo C, Ltd, Osaka, Japan). Finally, the reaction mixtures were reverse transcribed at 37°C for 60 min, heated at 95°C for 5 min, and stored at −20°C until use. PCR was performed in a 25 μl final reaction volume containing 20 mM Tris-HCl (pH 8.3); 50 mM KCl; 2.5 mM MgCl2; 200 μM each of dNTP; 0.5 μM of each primer (sense 5′-GGC ATC TGA GAC CAG TGA GAA-3′, antisense 5′-CTG TATG AGT CCT GGT GTG GG-3′); 0.25 units of polymerase (AmpliTaq Gold with GeneAmp, Roche Molecular Systems), and 3 μl of cDNA from each RT reaction (target cDNA with unknown concentration and competitive cDNAs with known concentrations). The amplification was done with a DNA thermal cycler (Perkin Elmer GeneAmp, PCR System 2400, Branchburg, NJ, USA) under the following conditions: hot start at 95°C for 9 min, denaturing at 95°C for 1 min, primer annealing at 64°C for 1 min, and chain elongation at 72°C for 2 min, through 35 consecutive cycles. PCR of competitor RNA yields a band of 221 base pairs (bp), while PCR of sample RNA yields a band of 281 bp. When the expected band was invisible, second round PCR was performed with the nested primers: sense 5′-GGC ATC TGA GAC CAG TGA GAA-3′ and antisense 5′-TGG GTC TTC AGG TGG TCG GAC-3′ in 25 μl reaction solution containing 2 μl of the first round PCR product. After amplification, 8 μl of each CR product was separated by electrophoresis in 2% agarose gel containing 0.5 μg/ml ethidium bromide. Gels were visualized and photographed under UV, and pictures were digitalized using Digital Image System FILE DF-20 scanner (FujiFilm, Osaka, Japan). WT1-specific PCR products from samples were quantified with Quantity-One software (PDI, Osaka, Japan) by comparison with the products obtained from competitor RNA.

Cytotoxicity assay

PBMNC cytotoxicity was assessed by flow cytometry according to Chang’s method26 with slight modification. Briefly, an appropriate number of K562 target cells were pelleted and resuspended in complete medium at a density of 1 × 106 cells/ml. After dispensing 10 μl of 3 mM Dio working solution into each tube, 1 ml of target cell suspension was added forcefully to disperse Dio. Cells were incubated at 37°C for 30 min, washed twice with phosphate buffer saline, and then resuspended in the medium at a density of 1 × 106 cells/ml. The cultured PBMNC (effectors) were added into two tubes to yield effector-to-target (E:T) ratios of 10:1 and 20:1, and into an effector control tube. Cells were pelleted and resuspended in 130 μl of medium. Dio-stained target cells were added to each of the E:T ratio tubes and to a separate target control tube, centrifuged at 1300 r.p.m. for 30 s, and then incubated at 37°C for 24 h. Before the end of incubation, 130 μl of PI working solution was added to the cocultured cells and incubated for another 30 min. Then, cytotoxicity was assessed using EPICS Profile II (Coulter, Hialeah, FL, USA). Dio-labeled target cells emitted green fluorescence (FL1), and PI-stained cells far red fluorescence (FL3). ‘Back gating’ of Dio stained K562 cells yielded a single parameter histogram (FL3), from which percent lysis could directly be obtained.26 The percent lysis of control target cells, that is spontaneous death of K562 cells, was taken as background. The background was subtracted from the percent lysis of the cocultured cells, and then the product was considered as specific percent cytotoxicity of PBMNC. Increased cytotoxicity was defined as an increase of at least 5% in cytolysis of K562 cells at the E:T ratio of 20:1 compared with that of the control culture.

Data are expressed as mean ± standard deviation (s.d.). Statistical analysis was performed by using the Student’s t-test and the Mann–Whitney U test. P values less than 0.05 were considered statistically significant.

Results

Effects of IL-12 on PBMNC cytotoxicity against K562 cells

The optimal activation time for PBMNC cytotoxicity by IL-12 was 3 days, and the optimal concentration of IL-12 was 100 units/ml (Figure 1). Under this condition, exposure of normal PBMNC to IL-12 significantly increased their cytotoxicity (Table 2). PBMNC from 30 patients with leukemia or MDS were tested next to see whether these cells could be activated by IL-12. In all 30 patients, the cytotoxicities of non-IL-12-treated PBMNC were 7.5 ± 4.9% and 13.4 ± 9.3% at the E:T ratios of 10:1 and 20:1, respectively, which were significantly lower than that of normal subjects (13.3 ± 4.8% and 25.7 ± 8.4%, P = 0.019 and P = 0.008 by the Student’s t-test, respectively). Following the 3-day IL-12 treatment, they significantly increased to 18.5 ± 12.5% and 30.6 ± 17.9%, respectively (P = 0.0004 and P = 0.0002 by the Student’s paired t-test, respectively), nearly corresponding to the levels of normal subjects (28.8 ± 9.0% and 41.8 ± 6.0%, respectively). The enhanced cytotoxicity was significantly different between five AML patients in CR (25.3 ± 15.0% and 39.5 ± 20.3% at the E:T ratios of 10:1 and 20:1, respectively) and seven AML patients in non-CR (10.7 ± 7.8% and 17.8 ± 13.6%; P = 0.02 and P = 0.02, respectively). As shown in Table 3, the cytotoxicity was increased in all six CML patients, 11 of 12 MDS patients, four of five AML patients in CR but in only three of seven AML patients in non-CR. The results indicated that IL-12 could enhance significantly the cytotoxicity of PBMNC from most patients with leukemia or MDS.

Figure 1
figure1

 Cytotoxicity of PBMNC from a healthy volunteer against K562. PBMNC (5 × 105 cells/ml) were incubated with different doses of IL-12 for 4, 24, 72 and 120 h. After the indicated times, PBMNC were tested for their cytotoxicity against K562 cells at an E:T ratio of 10:1. Y axis is percent lysis of K562 cells by PBMNC. The figure is a representative one of three experiments performed with similar results.

Table 2  Cytotoxicity against K562 and WT1 gene expression in PBMNC of healthy volunteers
Table 3  Effect of IL-12 (100 U/ml) on cytotoxicity of patients’ PBMNC against K562

WT1 mRNA expression in K562 cells and PBMNC from healthy volunteers

WT1 mRNA quantified by competitive RT-PCR was more than 107 copies/μg of total RNA in K562 cells (Figure 2a) and less than 103 copies/μg of total RNA in PBMNC from healthy volunteers (Figure 2b).

Figure 2
figure2

 Competitive RT-PCR of WT1 mRNA. PCR fragments were separated on 2% ethidium bromide-stained agarose gel. The amounts of competitor RNA are 107, 106, 105, 104, 103 and 0 copies/μg of total RNA (lanes 1–6, respectively). WT1 mRNA was more than 107 copies/μg of total RNA in K562 cells (a) and less than 103 copies/μg of total RNA in PBMNC from healthy subjects (b). IL-12 alone showed no direct effect on WT1 mRNA expression in K562 cells (c: IL-12 (−), d: IL-12 (+)). In a CMML patient, WT1 mRNA reduced from 106 copies/μg of total RNA (e) to 104 copies/μg of total RNA (f) after the 3-day IL-12 treatment.

Effects of IL-12 on WT1 mRNA expression in K562 cells and in PBMNC from patients

K562 cells were cultured with or without 100 units/mL of IL-12 for 3 days. IL-12 treatment had no direct effect on WT1 mRNA expression in K562 cells (Figure 2c, d). Then, we cocultured 5 × 105 PBMNC from a healthy volunteer and 5 × 103 K562 cells/ml with or without IL-12 for 3 days. The WT1 mRNA expression was 104.5 copies/μg of total RNA in the non-IL-12-treated cocultured cells and reduced to 104.0 copies/μg in the IL-12-treated cocultured cells, accompanied by an increase in cytotoxicity of PBMNC of the same source from 18.7% to 36.8% at the E:T ratio of 20:1 after the 3-day IL-12 treatment.

For clinical samples, as shown in Figure 3, in six CML patients, mean WT1 mRNA was 104.8 (range, 104.0–105.0) copies/μg of total RNA without the treatment, and was reduced to 104.2 (range, 103.5–104.5) copies/μg after the 3-day IL-12 treatment (P = 0.04, the Mann–Whitney U test). In 12 MDS patients, mean WT1 mRNA was 105.4 (range, 103.5–106.0) copies/μg without the treatment, and was reduced to 104.8 (range, 103.0–105.5) copies/μg after the 3-day IL-12 treatment (P = 0.008, the Mann–Whitney U test). In one CMML patient, WT1 mRNA was reduced from 106.0 to 104.0 copies/μg of total RNA after the 3-day IL-12 treatment (Figure 2e, f). In five AML patients in CR stage, WT1 mRNA was reduced from 105.0 (range, 103.0–105.5) to 104.2 (range, 102.5–104.5) copies/μg after the 3-day IL-12 treatment (P = 0.04). Among seven AML patients in non-CR, however, the 3-day IL-12 treatment reduced WT1 mRNA in only two patients (Patients Nos 24 and 25 in Table 1). Both of them showed increased cytotoxicity despite the presence of peripheral leukemia blasts, (18% and 27%, respectively). In the other five patients with more than 30% of peripheral leukemia blasts, WT1 mRNA did not change after the 3-day IL-12 treatment. The data indicated that IL-12 treatment diminished the amount of WT1 mRNA in most leukemia and MDS patients whose PBMNC contained less than 30% of leukemia blasts.

Figure 3
figure3

 WT1 mRNA expression in PBMNC with or without IL-12 treatment in 30 patients with leukemia or MDS. IL-12 treatment reduced WT1 mRNA in five of six CML in chronic phase, nine of 12 MDS, all of five AML in CR stage, and in two of seven AML in non-CR stage.

Linear regression analysis showed a weak but significant correlation (r = 0.439, P = 0.015) between the increases in the PBMNC cytotoxicity against K562 (at the E:T ratio of 20:1) and the decreases in WT1 mRNA following the 3-day IL-12 treatment (Figure 4).

Figure 4
figure4

 Correlation between the increases in cytotoxicity (at the E:T ratio of 20:1) and the decreases in WT1 gene expression after the IL-12 treatment. Cytotoxicity was assessed by flow cytometry and decreases in WT1 mRNA expression were detected by competitive RT-PCR. Correlation coefficient r = 0.439, P = 0.015.

Discussion

Despite considerable advances in therapy of hematological malignancies, minimal residual disease (MRD) is still a major problem, and immunotherapy has been one of the long-sought goals for the treatment of MRD. The evidence that IL-12 is capable of regulating many activities in both cellular and humoral immune responses,1234567891011121314 has made IL-12 an attractive agent for immunological manipulation on MRD in patients with hematological malignancies.

Most previous works used flow cytometer-sorted NK cells or non-adherent mononuclear cells of patients6 or PBMNC of healthy volunteers5 to examine the effects of IL-12. In the present study, however, total PBMNC including NK cells, lymphocytes, monocytes and even leukemia blasts were used, since we believed that total PBMNC would reflect the in vivo conditions in patients more. PBMNC cytotoxicity was significantly lower in the patients than in normal subjects. IL-12 treatment increased PBMNC cytotoxicity against K562 cells in all six CML patients, 11 of 12 MDS (three RA, eight-RAEB and one CMML) and seven of 12 AML. This finding is different from that of Ogata et al,6 who showed that treatment of non-adherent PBMNC (1.5 × 106 cells/ml) with IL-12 alone (2.5 units/ml) for 18 h did not enhance the lymphocyte cytotoxicity in all three RAEB and two RAEB-in-transformation patients, although the combination of IL-12 and IL-2 showed some effect. The discrepancy between theirs and our results could be explained by the differences in cell population, cell density in culture, doses of IL-12 and treatment time. We cultured the total PBMNC including cytotoxic T lymphocytes, monocytes and NK cells at the density of 5 × 105 cells/ml with 100 units/ml of IL-12 for 3 days. In this experiment system, IL-12 would activate not only NK cells but also cytotoxic T lymphocytes and monocytes, and produce INF-γ, TNF-α and other cytolytic components such as perforin, serine esterase and TIA-1 during the 3-day treatment.

In the present study, we used WT1 mRNA as a marker for MRD. Although it is controversial whether WT1 mRNA expression could be used as a reliable marker for MRD in leukemia,27 the majority of published works have reported the usefulness of this gene expression in the prognosis of leukemia and MDS.1922232428 Inoue and his collogues28 showed that WT1 mRNA expression in normal bone marrow cells was at either very low or undetectable levels, and that MRD detected by quantitation of WT1 gene expression was comparable to MRD simultaneously measured by RT-PCR using primers for specific DNA markers (PML/RAR-α for M3, AML1/ETO for M2, and bcr/abl for CML) and to the number of leukemia blasts as well.1923 In our study, the IL-12 treatment reduced WT1 mRNA expression in the coculture of normal PBMNC and K562 cells, and increased the cytotoxicity of PBMNC of the same normal donor against K562 cells. The results suggested that WT1 mRNA expression could be used for the measurement of residual amounts of K562 cells. Additionally, there was a significant correlation between the decrease in WT1 mRNA expression and the increase in cytotoxicity of PBMNC in patients with leukemia or MDS, which also implied that WT1 expression could be used for the measurement of residual amounts of leukemia cells in these patients. However, it remains to be ascertained whether the diminution in WT1 gene expression following the IL-12 treatment was really due to a true decrease in MRD or whether some other mechanism may be responsible for the effect. Further study is certainly required to clarify this.

Severe toxicity has been a major obstacle for the intravenous use of IL-12 in patients with advanced malignancies in a phase II study of IL-12.29 However, by subcutaneous administration, IL-12 has been well tolerated and showed promising antitumor effects in patients with cutaneous T cell lymphoma15 and metastatic renal cell carcinoma.161718 Therefore, our study might be of considerable clinical benefit, if IL-12 is also appropriately applied in the treatment of MRD in hematological malignancies. Besides, ex vivo purging before autologous stem cell transplantation by IL-12 or its combination usage with other cytokines such as IL-2 may offer a new approach in the treatment of hematological malignancies.

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Acknowledgements

We thank Dr M Kobayashi (Center Molecular Biology and Cytogenetics, SRL, Japan) for his kind technical advice about competitive RT-PCR. We also thank Dr Quang-Kim Tran for his helpful proofreading of the manuscript.

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Correspondence to K Ohnishi.

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Pan, L., Ohnishi, K., Zhang, W. et al. In vitro IL-12 treatment of peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes: increase in cytotoxicity and reduction in WT1 gene expression. Leukemia 14, 1634–1641 (2000) doi:10.1038/sj.leu.2401872

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Keywords

  • IL-12
  • cytotoxicity
  • competitive RT-PCR
  • WT1 mRNA
  • leukemia
  • MDS

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