Restoration of Th1 Cytokine Synthesis by T Cells of Patients with Chronic Myelogenous Leukemia in Cytogenetic and Hematologic Remission with Interferon-a

Chronic myelogenous leukemia (CML) is a disorder of the hematopoietic stem cell that results in malignant expansion of myeloid cells with a cytogenetic abnormality, the translocation between chromosomes 9 and 22 known as the Philadelphia chromosome. Treatment with IFN-a has proven to be an effective therapy, inducing cytogenetic remission in CML patients. However, it is unknown whether IFN-a can restore normal immune function for patients who achieve a complete cytogenetic remission. To address this question, we used a method of intracellular staining and flow cytometric analysis to ascribe the syntheses of Th1 or Th2 cytokines to T-cell subsets of patients in chronic, in accelerated, and in blast crisis phases as well as patients who had achieved a complete cytogenetic remission with IFN-a. We assessed the cytoplasmic synthesis of cytokine in phorbol ester (phorbol 12-myristate 13-acetate)-activated CD41 and CD81 T-cell subsets of 81 patients with various stages of CML and 21 normal controls. The percentages of CD41 and CD81 T cells from patients in chronic, in accelerated, and in blast crisis phases that synthesized Th1 cytokines interleukin (IL)-2, IFN-g, and tumor necrosis factor-a were significantly lower than those of remission patients and normal controls. Conversely, the percentages of CD41 and CD81 T cells of patients in chronic, in accelerated, and in blast crisis phases of CML preferentially synthesized the Th2 cytokine IL-10. Patients who achieved a durable complete cytogenetic remission for >2 years without maintenance IFN-a therapy restored their preference for a Th1 cytokine profile that is necessary for efficient cytotoxic T-cell function. INTRODUCTION CML is a malignancy of the hematopoietic stem cells resulting in the clonal expansion of myeloid cells and their progenitors (reviewed in Refs. 1 and 2). At least 90% of the malignant cells in CML are characterized by a cytogenetic abnormality, known as the Philadelphia chromosome (Ph1; Ref. 3), the result of a reciprocal translocation between chromosomes 9(q34) and 22(q11) (Ref. 4). Patients with CML present in the Chronic phase characterized by a relatively indolent, benign stage with the differentiation of precursors into functional, mature hematopoietic cells. The disease can be either biphasic or triphasic, because some patients proceed into a less well-defined accelerated phase wherein the myeloid cells begin to stop maturing into granulocytes and instead become arrested at the level of the immature blast (5). Patients in ACC phase have 15–30% blasts and promyelocytes and #20% basophils in the blood or bone marrow. The natural course of CML is a transformation that closely resembles an acute leukemia, known as the BC phase, during which blasts of both lymphoid and myeloid origin make up at least 30% of the WBCs in bone marrow or blood (6). Transformation is often associated with karyotypic evolution (7). Patients become refractory to chemotherapy (8), and survival for patients in ACC/BC is on the order of only 3–12 months (6). Conventional treatment of CML with chemotherapeutic agents normalizes the WBC count of patients but indiscriminately kills both the leukemia clone and normal cells (9); the chemotherapeutic agents are often considered palliative because they do not delay the onset of BC (10). Treatment with IFN-a, on the other hand, not only reduces the WBC count but also reduces the neoplastic, Ph1 cells (11, 12). Possible mechanisms of action by IFN-a include the reduction of tumor burden (11, 12), through the down-regulation of BCR/ABL mRNA and P210 (BCR/ABL) protein (13), regulation of tumor necrosis factor-related apoptosis-inducing ligand-mediated T-cell cytotoxicity (14), suppression of the chemotactic protein IL-8 (15), induction of the inflammatory factors IL-1 receptor antagonist (16) and soluble receptor p55 of tumor necrosis factor (17), and the regulation of the cytokine network by suppressing Th2-like cytokine responses (18) that are capable of suppressing normal T-cell proliferation (19). Although IFN-a is known to inhibit the growth of bone marrow progenitors and to restore adherence properties to Received 12/19/99; revised 2/11/00; accepted 2/11/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Division of Pathology and Laboratory Medicine, Box 7, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-3559; Fax: (713) 792-4296; E-mail: [email protected]. 2 The abbreviations used are: CML, chronic myelogenous leukemia; ACC, accelerated (phase of CML); BC, blast crisis; IL, interleukin; CCR, complete cytogenetic remission; Late-CCR, long-term remissions; Early-CCR, early remissions; PMA, phorbol 12-myristate 13-acetate; PE, phycoerythrin; TNF, tumor necrosis factor. 1671 Vol. 6, 1671–1677, May 2000 Clinical Cancer Research Research. on November 11, 2017. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from stroma in CML (20, 21), little is known of the effect of IFN-a therapy on the synthesis of Th1 and Th2 cytokines by T cells. Because Th1 and Th2 cytokines are important mediators of the host cellular immunity (22), the contribution of individual T-cell subsets with respect to cytokine synthesis warrants investigation. A shift from a Th1 to a Th2 cytokine profile is widely accepted as a prognosticator of disease progression in individuals with neoplasm (23, 24) or infection (25–28). We expected to find a Th2 cytokine profile in CML patients in Chronic and ACC/BC phases of disease and a Th1 cytokine profile of patients in complete cytogenetic remission after treatment with IFN-a. Furthermore, we hypothesized that CCR patients would have a Th1 cytokine profile comparable with normal controls. To test this hypothesis, we used a method of flow cytometry that permitted the evaluation of Th1 and Th2 cytokine syntheses in the cytoplasm of CD41 and CD81 T cells of CML patients at different stages of disease and compared their responses to patients who had achieved a CCR with IFN-a therapy. MATERIALS AND METHODS Staging and Classification of CML Patients A medical history including prior treatments for CML was taken from all patients participating in this study. A physical examination, including performance status and staging of disease, was done for each patient. The breakdown of the 81 patients by stage of disease is shown in Table 1. Patients with a diagnosis of Ph1 CML who had received IFN-a as their primary treatment or chemotherapy at the M. D. Anderson Cancer Center were included in this analysis. Treatment response criteria were defined previously (29, 30). BC was defined as .30% blasts in blood or bone marrow; ACC phase consisted of patients with blasts $15% and #30% or .30% blasts and promyelocytes and $20% basophils in the blood or bone marrow; the Chronic or benign phase of CML comprised of all other patients who did not fit the above clinical disease stages. Patients in whom a CCR was induced with IFN-a were subdivided into Late-CCR and Early-CCR. Late-CCR patients were those who had a continuous CCR for .2 years and in whom IFN-a therapy was discontinued, whereas Early-CCR patients had a remission duration of ,2 years and required maintenance IFN-a therapy. Study Population The study population consisted of 81 CML patients who were assigned to a disease stage based on cytogenetic analysis of bone marrow and clinical assessment (30). The clinical stages of the CML patients included 15 patients in the Chronic phase (Chronic), 11 in accelerated or blast crisis phases (ACC/BC), 40 in Early-CCR, and 15 in Late-CCR (Table 1). Intracellular Cytokine Synthesis by T-Cell Subsets after Activation with PMA Each subject provided 5 ml of blood in EDTA (Vacutainer; Becton Dickinson, San Jose, CA) for a complete blood count, leukocyte differential analysis, and determination of lymphocyte phenotypes by flow cytometry. Another 10 ml of blood in heparin was obtained for cytokine studies. Informed consent was obtained from each subject with approval for the study from the Human Experimentation Committee of the University of Texas M. D. Anderson Cancer Center. Activation of T Cells by PMA. Blood samples from patients with a WBC count .15 3 10/ml were diluted to 15 3 10/ml to bring the WBC to within normal range. Intracellular cytokine synthesis was conducted in three stages as described previously (24). Briefly, 25 ng of PMA were added to 1 ml of whole blood, and the mixture was incubated for 4 h at 37°C in the presence of 1 mg of ionomycin and 10 mg of brefeldin-A [a nontoxic but potent inhibitor of intracellular transport (31)]. All reagents were purchased from Sigma Chemical Co.(St. Louis, MO). Staining of the Activated T cells. The PMA-activated cells were stained for the presence of cytoplasmic cytokines as described previously (24) using a panel of cytokine-specific monoclonal antibodies conjugated with PE to detect one of the following cytokines: IL-2, IL-10, IFN-g, and TNF-a. Additionally, samples were reacted with anti-CD8 monoclonal antibody conjugated with FITC and anti-CD3 monoclonal antibody to delineate CD81 and CD82 (or CD41) T cells. The percentage of cells that expressed the early activation antigen, CD69 (32), was indicative of lymphocyte activation by PMA. All monoclonal antibody reagents except for IL-10 were purchased from Becton Dickinson Immunocytometry Systems, Inc. (Mountain View, CA); anti-IL-10 conjugated with PE was purchased from Caltag (Burlingame, CA). Stained cell preparations were fixed Table 1 WBC counts and percentages of lymphocytes of CML patients and normal controls Study group No. per group WBC 3 10/ml % lymphocytes Lymphocytes count/ml Median Range Median Range Median Range Chronic 15 25.8 7.8–88.6 10.6 2.3–23.1 2434 1288–4030 ACC/BC 11 7.1 2.2–46.3 11.3 3.8–35.1 868 249–3102 Early-CCR 40 5.5 2.1–10.3 27.4 8.1–50.9 1380 433–2675 Late-CCR 15 6.8 3.8–30.1 29.4 3.7–33.6 1795 1098–4247 Controls 21 6.2 4.3–10.0 30.9 19.5–43.5 1871 1104–3175 a–f Data are presented as median and ranges for each parameter. Statistical analyses were among groups and between groups: a Chronic versus other groups, P , 0.01; b Chronic, ACC/BC versus Early-CCR, Late-CCR, and Normal Control, P , 0.001; c Chronic versus other groups, P , 0.01; d ACC/BC versus Early-CCR, Late-CCR, and Normal Control, P , 0.01; e Early-CCR versus Normal Control, P 5 0.02; f Early-CCR versus Late-CCR and Normal, P , 0.01. 1672 Th1 Cytokine Synthesis by CML Patients in Remission Research. on November 11, 2017. © 2000 American Association for Cancer clincancerres.aacrjournals.org Downloaded from in a solution of 1% paraformaldehyde and analyzed immediately or stored at 4°C for analysis within 24 h. Determination of Cytokines in the Cytoplasm of Activated T Cells by Flow Cytometry Cell preparations of PMA-activated cells were analyzed with the FACSCalibur flow cytometer (Becton Dickinson) using the CellQuest software. Briefly, 10,000 events were collected based on side scatter and anti-CD3 reactivity. Immunoglobulin isotype controls were used to verify the staining specificity of the anti-cytokine reagents and to set markers delineating positive and negative populations. Cytokine synthesis by the two T-cell subsets (CD31/CD81/CD42 and CD31/CD82/ CD41) were measured based on the reactivity of the lymphocytes with anti-CD8. Anti-CD69-PE was used to identify activated T cells after exposure to PMA. Statistical Analysis The mean 6 SE percentages of T-cell subsets synthesizing the cytokines of interest were obtained for each sample. Data are presented as histograms of the mean 6 SE percentages of CD81 and CD41 T cells/ml synthesizing a cytokine. Statistical differences between study groups with respect to the percentages of T cells synthesizing cytokines were determined by the Mann-Whitney test.