Jürgen Brieger

Lactate dehydrogenase-release assay: A reliable, nonradioactive technique for analysis of cytotoxic lymphocyte-mediated lytic activity against blasts from acute myelocytic leukemia

Treatment of patients in remission of acute myelocytic leukemia using immunotherapy with interleukin 2 is a new approach to prolonging remission duration in this disease. As an important mechanism for the pathophysiology of eradication of residual myelocytic blast populations, activation of cytotoxic effector lymphocytes has frequently been discussed. However, the associated immunological research has been complicated to some extent, because in conventional chromium 51-release assays, blast cells frequently fail to incorporate sufficient amounts of51Cr and/or spontaneously release high amounts of51Cr. Recently, we established a culture system which promotes the out-growth of cytotoxic T lymphocytes in bone marrow-derived mononuclear cells cultured in IL-2. To study cytotoxicity and the responsible mechanisms of the obtained T-cell lines and clones, we modified a previously described cytotoxicity assay, based on the release of lactate dehydrogenase (LDH-release assay) for use in cryopreserved blasts obtained from the bone marrow of patients with acute myelocytic leukemia. Using this assay, we were able to detect cytotoxicity of IL-2-activated peripheral blood lymphocytes from three healthy controls against a number of blast samples obtained from the bone marrow of patients with AML (up to more than 40% lysis at an effector target cell ratio of 20∶1). However, a minority of AML blasts seem to be resistant to lysis by IL-2-activated lymphocytes. In bone marrow-derived T-cell lines from patients with AML we detected lytic activity against autologous blasts in three of seven cases tested by LDH release, ranging from 29 to 63% at an effector target ratio of 10∶1. Additionally, T-cell clones with different phenotypes were established which were able to mediate cytotoxicity against autologous blast cells. Thus, cytotoxicity against freshly isolated blasts from patients with acute myelocytic leukemia can be analyzed reliably, reproducibly, and without the use of isotopes by the LDH-release assay.

Bone marrow-derived T-cell clones obtained from untreated acute myelocytic leukemia exhibit blast directed autologous cytotoxicity

The eradication of minimal residual blast populations by activation of autologous cytotoxic cells with interleukin 2 (IL-2) is a new promising tool in the treatment of acute myelocytic leukemia (AML). However, the immunological effector cells are not yet clearly defined. The present study was designed to investigate the presence of cytotoxic precursor cells in active AML and to identify phenotypical and functional characteristics of autologous anti-leukemic cytotoxic effector cells. For this purpose, mononuclear cells (MNC) containing at least 70% leukemic blasts were isolated from bone marrow of untreated AML and cultured in the presence of 3000 IU/ml recombinant IL-2 (rIL-2) for 6-8 weeks. Under these conditions, T-cells were selected in the bone marrow cultures and overgrew the leukemic blasts. The resulting T-cell populations were cloned by limiting dilution and the clones obtained were characterized for their phenotypical and functional patterns. Totally, cloning resulted in 68 clones and a few cell lines. The clonality was verified by RT PCR analysis of TCR V beta gene expression. All clones obtained stained positive for CD2, CD3, DR and CD56. The vast majority (68%) of T-cell clones/lines was CD4+, a few clones expressed CD8 (19%) or CD4 and CD8, and four clones were of TCR gamma delta origin. Seven of 15 clones tested, including three CD4+, two CD8+ and two TCR gamma delta(+)-clones were found to be cytotoxic against autologous leukemic blast cells. All except one clone expressed oncolytic activities against allogeneic blasts too. One of the TCR gamma delta(+)-clones demonstrated NK activity by lysis of K562 targets. The majority of the T-cell-clones released IL-2, IL-8, TNF-alpha, GM-CSF but only a few IFN gamma and expressed high levels of mRNA for IL-2, TGF-beta and IL-10. None of the clones was found to produce IL-3, IL-4, IL-7 and TNF-beta. The data provide evidence of the existence of T-cell precursors in untreated AML bone marrow differentiating to cytotoxic cells with activity against autologous and allogeneic AML blast cells.

Establishment and characterization of a new, factor-independent acute myeloid leukemia line designated Ei501

We established a factor-independent acute myeloid leukemia cell line, designated Ei501. The line has been growing in RPMI 1640 media for 18 months and can be maintained without addition of growth factors. Ei501 is positive for myeloperoxidase and negative for esterase and PAS. Cytogenetic analysis revealed the FAB M3 associated t(15;17) translocation and a translocation of the chromosomes 7 and 8: 46 XX, −7, +t(7;8)(q32;q13), t(15;17)(q22;q12). This karyotype was confirmed by fluorescence in situ hybridization. Ei501 cells express AML-associated surface markers such as CD13, CD33 and CD38. Although 42% of the patient’s blast cells were CD34-positive, the line lacks surface expression of CD34. Furthermore the line has a number of characteristics which are detectable in blasts from AML patients, such as surface adhesion molecules, cytokines such as TGF-β, cytokine receptors such as the IL-2 receptor β and γ chains or the IL-4 receptor and the genes for the transcription factor wt- 1 (Wilms’ tumor gene) and for the proto-oncogene bcl-2, both shown to be present in the majority of patients with AML. Additionally the line can be used as target in cytotoxicity assays using IL-2 activated cytotoxic lymphocytes as effector cells. In conclusion, besides a rare karyotype the Ei501 cell line has several features common in AML, and may therefore be used as a model to study pathogenetic mechanisms in acute myeloid leukemia.

In vivo regulation of transforming growth factor β1 transcription by immunotherapy : interleukin-2 impairs interferon-α-stimulated increase in steady-state mRNA levels of transforming growth factor β1

Recombinant interleukin-2 (rIL-2) in combination with recombinant interferon α (rIFNα) has been shown to mediate significant antitumoral effects in some patients with advanced renal cell cancer or malignant melanoma. The therapeutic effects may be partially modulated by secondarily induced cytokines, especially with regard to in vivo lymphocyte activation. To investigate possible negative effects on lymphocyte activation during immunotherapy, we designed a study on transcription of transforming growth factor β1 (TGFβ1), a known inhibitor of lymphocyte function, in patients undergoing treatment with daily alternating administration of rIFNα and rIL-2. Here we present data on gene expression of TGFβ1. Kinetic mRNA studies revealed an increase of TGFβ1 mRNA in peripheral mononuclear cells 12 h after subcutaneous injection of rIFNα. The following intravenous rIL-2 administration significantly decreased the amounts of TGFβ1-specific mRNA. In contrast to the effect of the first dose, subsequent application of rIFNα did not enhance TGFβ gene expression during rIFNα/IL-2 therapy. The diminished TGFβ1 gene expression returned to pretreatment levels 1–7 days after the last rIL-2 administration. When IL-2 expression was studied, increase in IL-2 mRNA was concomitant with a decrease in TGFβ1 transcripts. Our results indicate a complex regulatory effect on secondarily induced cytokines such as TGFβ1 by immunotherapeutic approaches. The rIL-2-mediated down-regulation of increased TGFβ1 steady-staty-state mRNA levels following rIFNα may represent a positive immune regulatory effect on cytotoxic cells. Furthermore this effect may modulate proliferation of neoplastic tissues.

AML blasts variably express interleukin 2 receptor α, β or γ chains without measurable effects on proliferation, cytokine message expression or surface expression of adhesion molecules upon stimulation with interleukin 2

Preliminary clinical studies including interleukin 2 (IL-2) in chemotherapy strategies for treatment of acute myeloblastic leukemia (AML) suggest that IL-2 may improve the disease-free survival of the patients. Because of reports showing interleukin 2 receptor expression on AML blasts, it is important to know whether IL-2 may directly influence these leukemic cells. In initial studies using flow cytometry to analyze surface expression of interleukin 2 receptors (IL-2R), we found expression of the IL-2R alpha chain on blast cells in 26% and of the IL-2R beta-chain in 81% of the patients. To confirm these results at the transcriptional level, we studied the expression of RNA of the IL-2R alpha, beta, and gamma chains by RT-PCR in the bone marrow of 38 newly diagnosed patients with AML and in three AML-derived cell lines. RNA of the alpha chain was detectable in 11/38 patients, the beta chain in 10/38 patients and the gamma chain in 30/38 patients with AML. Blast cells obtained from colonies growing in semisolid media expressed mRNA for IL-2R. RNA for all three IL-2R chains was expressed in lines KG1, HEL 92.1.7 and K562. In comparison with unstimulated cell lines, incubation of the three lines with various amounts of IL-2 over 3 and 14 days did not increase their growth or change message expression of IL-2R, IL-10, and TGF-beta and surface expression of the adhesion molecules CD 11, CD 18, CD 29 and CD 54. In conclusion, despite expression of IL-2 receptors, AML blasts do not respond to IL-2 by proliferation, message expression for various cytokine genes and surface expression of cellular adhesion molecules.

IL-2 Receptor (IL-2R) Alpha, Beta, and Gamma Chains Expressed on Blasts of Acute Myelocytic Leukemia May Not Be Functional

Preliminary clinical studies including IL-2 in chemotherapeutic strategies for treatment of acute myelocytic leukemia suggest that IL-2 may improve the overall outcome of the patients [1, 2]. To date, the mechanisms of eradication of blast cells are still unclear. Cytotoxic T and NK cells or secondary cytokines released after administration of IL-2 have been discussed to contribute to the elimination of leukemic cells [3–5]. However, it is important to know, whether IL-2 may directly influence AML blasts [6]. In initial sudies, using flow cytometry, we found expression of considerable levels of the IL-2R alpha chain on blast cells in a low proportion of patients with AML. However, the IL-2R beta chain was more frequently expressed ranging from 0% to 98%. To confirm these results on a transcriptional level, we studied the expression of RNA of the IL-2R α, β, and γ chains by RT PCR in the bone marrow of 39 newly diagnosed patients with AML and in three AML derived cell lines. RNA for all three IL-2R chains was expressed in lines KG1, HEL 92.1.7 and K562. Surface expression of the IL-2R β chain was observed in all three lines, but of the α chain only in KG1 cells. In comparison with unstimulated cell lines incubation of the three lines with various amounts of IL-2 over 3 and 14 days did not influence their growth, measured by cell numbers and 3H-thymidin incorporation. RNA for the α chain was detectable in 12/39 patients, of the β chain in 10/39 patients and the γ chain in 29/39 patients with AML. Altogether the data suggest that the IL-2R expressed on AML blast cells may be incomplete and not functional. In future studies, functional properties of IL-2 R on blast cells obtained from newly diagnosed patients with AML will have to be investigated.

Detection of the Wilms Tumor Gene (wt-1) mRNA in Complete Remission of AML Frequently Precedes Relapse

Leukemic blast cells taken from 83 patients with AML at time of diagnosis and 20 patients during follow-up in complete remission were examined for the expression of wt-1 mRNA. For this purpose blast cells were isolated from bone marrow or peripheral blood and total RNA was extracted. The wt-1 transcription was subsequently studied via RT-PCR. Mononuclear cells and bone marrow from healthy persons were used as controls. Wilms’ tumor mRNA was detectable in 67 out of 83 cases of AML (81%). None of the 13 healthy controls expressed wt-1. After achieving cytological Complete Remission (CR) 14/20 patients studied lost wt-1 expression. In 7/8 patients in CR persistence or reappearance of wt-1 expression preceded relapse of the disease. Response to therapy and survival did not correlate with wt-1 mRNA expression in newly diagnosed AML before therapy. Our data show that expression of wt-1 mRNA is widely spread in AML and may be a useful marker for detection of minimal residual disease (MRD) in acute leukemia. The follow-up data strongly suggest that analysis of wt-1 gene expression via PCR in CR may be a sensitive technique for the early detection of relapse. Analysis of wt-1 expression accompanying routine bone marrow aspiration may be useful to define the quality of remission and early prediction of relapse of the disease. Its relevance to outcome, follow-up, and detection of MRD, however, as well as its potential role in the pathogenesis of acute leukemia, remain to be confirmed by ongoing studies.

The Inhibition of Lymphokine Activated Killer Cell Activation Mediated by AML Culture Supernatants Might Be Due to Transforming Growth Factor Beta1

The cytotoxic activity of peripheral mononuclear cells (PMNC) from patients with acute myelogenous leukemia (AML) is usually reduced at time of diagnosis [1, 2]. Production and release of immunosuppressive cytokines by leukemic blast cells might be a cause for impaired cytotoxic activity and immunosurveillance of leukemic cells. Indeed, soluble but yet undefined factors secreted by AML blasts have been described to be reponsible for inhibited cytotoxic activity in AML patients [3, 4]. Transforming growth factor beta (TGF-β) has been reported to be a strong inhibitor of cytotoxic activity of lymphokine activated killer (LAK) cells [5–9]. In ovarian carcinoma, increased TGF-β secretion was shown to suppress various immune functions [10]. It may be suggested that TGF-β may be an important factor in AML, too, causing immunosuppression.

The Amplification of the Wilms Tumor Gene (wt-1) mRNA Using the Polymerase Chain Reaction Technique (PCR) May Enable Sensitive Detection of Small Blast Populations in AML

Leukemic cells from 52 patients with AML were examined for the expression of wt-1 mRNA. Blast cells were isolated from bone marrow or peripheral blood. Total RNA was extracted and wt-1 transcription was studied via RT-PCR. Mononuclear cells and bone marrow from healthy persons were used as controls. Cell surface antigens were determined using FACS-analysis. In dilution experiments the detection limit was assessed as 1 out of 10000 cells. Wilms’ tumor mRNA was detectable in 41 out of 52 cases of AML (79%). None of the 13 controls expressed wt-1. After chemotherapy, six out of eleven patients in complete remission (CR) lost wt-1 expression completely. The remaining five patients expressed wt-1 mRNA at the same or a lower level. The significance of wt-1 persistance for desease free survival will have to be defined.

Expression of CD7 and CD15 on Leukemic Blasts Are Prognostic Parameters in Patients with Acute Myelocytic Leukemia — Irrelevance of CD34

In order to investigate the clinical significance of surface markers in correlation to therapeutic outcome of induction therapy in acute myelocytic leukemia (AML), the blasts from 94 adult patients with AML were analyzed prospectively. We used a panel of 32 monoclonal antibodies reactive with normal lymphoid and myeloid cells at various stages of differentiation in a direct immunofluorescence technique. There was no strict correlation of antigen expression with the French-American-British (FAB) classification. All patients were treated with an intensive induction treatment. Staining by two antibodies had a prognostic value. The achievement of complete remission (CR) was significantly correlated with a high (> 50% of cells) expression of CD7 in de novo AML. All patients (8/39) with de novo AML and high expression of CD7 achieved CR, so far (p < 0.03). The high expression of CD15 was also correlated with the achievement of CR (p < 0.05). In contrast to recent reports, we found no correlation between CD34 surface expression and the prognosis of the disease. These results confirm earlier reports of antigenic heterogeneity in AML, and indicate that immunologically defined subgroups of AML patients which are of potential clinical significance can be identified.