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Imatinib mesylate suppresses cytokine synthesis by activated CD4 T cells of patients with chronic myelogenous leukemia 1Department of Hematopathology, The pandora braclet charms University of Texas MD Anderson Cancer Center, Houston, TX, USA2Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA3Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USACorrespondence: Dr JM Reuben, Department of Hematopathology, Unit 0054, Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA.

Top of pageAbstractAlthough imatinib mesylate (IM) is highly effective at inducing complete cytogenetic remission in patients with chronic myelogenous leukemia (CML), it is known to suppress T cell proliferation in vitro. As cytokines are required for T cell proliferation, we investigated the effects of IM on cytokine synthesis by T cells of CML patients by assessing cytokine synthesis by activated CD4+ and CD8+ T cells in vitro. The activation of T cells in the whole blood of IM treated patients (CML IM) with Staphylococcus enterotoxin B resulted in significantly lower percentages of CD4+ T cells that synthesized interleukin 2 (P=0.017), interferon gamma (P=0.010), and tumor necrosis factor alpha (P=0.009) than did the activated T cells of control subjects. The addition of exogenous IM to the cultures of peripheral blood mononuclear cells of CML IM patients reduced Th1 cytokine synthesis by the CD4+ T cells. Furthermore, IM therapy at clinical doses suppressed the tyrosine phosphorylation of ZAP70. These findings suggest that inhibition of ZAP70 signaling pathway and suppression of Th1 cytokine synthesis by CD4+ T cells required the presence of IM at the time of T cell activation through the T cell receptor.Keywords: chronic myelogenous leukemia, imatinib mesylate, cytokines, T cellsTop of pageIntroductionChronic myelogenous leukemia (CML) is a malignancy of the hematopoietic stem cells resulting in the clonal expansion of myeloid cells and their progenitors.1 At least 90% of cases have a cytogenetic abnormality known as the Philadelphia chromosome (Ph+), which results from a reciprocal translocation between chromosomes 9 (q34) and 22 (q11). CML patients typically present in a chronic phase of the disease, which is a relatively indolent stage when precursors are differentiating into functional, mature hematopoietic cells. Recently, imatinib mesylate (IM), also known as Gleevec (Novartis, Basel, Switzerland) or STI 571, an inhibitor of BCR ABL tyrosine kinase, has been shown to be a highly potent treatment for CML.2 IM induces complete cytogenetic remission (CCR) in 74% of newly diagnosed CML patients and is also active in CML patients previously treated with interferon alpha (IFN ), with as many as 45% of these latter CML patients achieving CCR.3, 4 IM specificity is achieved through its high affinity for the ATP binding pocket of specific target kinases, including Abelson kinase (c ABL), the stem cell factor receptor c kit, and the platelet derived growth factor receptor.5 This drug is well tolerated by patients with CML and other types of leukemia and does not produce the overt toxicity associated with conventional cancer therapy.Reuben et al6 and Guarini et al7 have shown that patients in chronic, accelerated, and blast crisis phases of CML have a Th2 cytokine profile, whereas patients who are in CCR after treatment with IFN have a Th1 cytokine profile. Although IM is highly effective at inducing CCR in CML patients, a drawback of IM therapy is that it can also inhibit cytokine production by the T cells of healthy subjects8 and suppress normal human T cell proliferation in vitro in a dose dependent manner.9 Cytokines, especially interleukin (IL) 2, are necessary for T cell proliferation, and together with interferon gamma (IFN ) and tumor necrosis factor alpha (TNF ), are necessary for an efficient T cell mediated immune response, whereas the Th2 cytokine IL 10 downregulates cell mediated immunity.10 As cytokines are essential mediators of host immunosurveillance mechanisms that are important for the eradication of CML, we investigate if IM inhibits cytokine synthesis by activated T cells of CML patients.Top of pageMaterials and methodsClinical staging and classification of patients with CMLThe patient population consisted of patients with Ph+ CML in CCR receiving either IM or IFN as their primary treatment at The University of Texas MD Anderson Cancer Center who had consented to participate in this study approved by the institution's Human Experimentation Committee. At the time of the study, the patients provided a medical history, including information about prior treatments for CML, and underwent a physical examination, including determination of the performance status, disease stage, and response to treatment, according to previously defined criteria.11 All patients had been in CCR for less than 2 years and were on maintenance therapy with either IM or IFN. Control subjects who self reported to be in good health and not on any immunomodulatory medication were also recruited to participate in the study. T cells activated with either PMA or SEB were stained for the presence of cytoplasmic cytokines, as previously described.6Intracellular cytokine synthesis by activated T cells in the absence of autologous plasmaPeripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll density gradient centrifugation and suspended in 'complete medium' consisting of RPMI 1640 (Whittaker Bioproduct, Walkersville, MD, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 U/ml penicillin (Whittaker Bioproduct), 100 g/ml streptomycin (Whittaker Bioproduct), and 2 mM glutamine (GIBCO, Grand Island, NY, USA). PBMCs (6 106) from each of 14 CML IM patients and 15 healthy controls were suspended in 1.5 ml of complete medium that was supplemented with SEB, anti CD28, and anti CD49d (each at a final concentration of 3 g/ml), and the cultures were incubated at 37 for 5 h. Brefeldin A was added to the cultures for the final 3 h of incubation. In addition, PBMC cultures were activated with a pan T cell activator, anti CD3, which was immobilized to the surface of a 24 well plate (Costar, Cambridge, MA, USA). Briefly, the bottom of each well of a 24 well plate was coated with 0.75 g of anti CD3 (Coulter Immunology, Hialeah, FL, USA), and the plate was incubated at 37 for 6 h. Thereafter, the plates were air dried in a laminar flow hood. Anti CD28 (1.5 g) (BD Biosciences, San Jose, CA, USA) and 106 PBMCs in 1 ml of complete medium were added to each well of a 24 well plate coated with anti CD3, and the plate was incubated at 37 for 16 h. In total, 10 g of brefeldin A was added to each PBMC culture for the last 3 h of the incubation period. Next, 6 106 activated PBMCs were harvested and reacted with monoclonal antibody reagents to detect the intracellular expression of cytokines, and the stained activated T cells were analyzed as described previously.6, 13Cytokine synthesis by anti CD3 activated T cells in the presence of IM or autologous plasma106 aliquots of PBMCs from CML IM patients (N=14) were suspended in medium containing anti CD28 and either 10% FBS plus 2.5 M IM or 10% autologous plasma. Next, the PBMCs were dispensed into the individual wells coated with an antibody to CD3, and the plate was incubated at 37 for 16 h. In total, 10 g of brefeldin A was added to each of the PBMC cultures for the last 3 h of the incubation period. Thereafter, the activated PBMCs were harvested for detection of intracellular cytokines, and the stained activated T cells were analyzed as previously described.6, 13Tyrosine phosphorylation of ZAP70 in PBMCs of CML patientsPBMCs from three CML IM patients were isolated and washed in PBS before the isolated pellet was stored at 140 PBMCs collected from individual CML IM patients before and after therapy with IM were lysed (on ice), and equal protein aliquots (30 g) were analyzed for p(Y319) ZAP70 and ZAP70 by immunoblotting and reprobed with antibody to ZAP70 (Cell Signaling Technology, Beverly, MA, USA) as a loading control. Jurkat cell lysate was used as a positive control. The clinical use of IM was the sole source of IM exposure in these PBMCs.Statistical analysisStatistical differences between the percentages of T cells charm bracelet pandora price synthesizing cytokines in the whole blood cultures of healthy controls, CML IFN patients, and CML IM patients in response to activation with PMA or SEB were examined by one pandora charms 2013 way analysis of variance, followed by Dunnett's T3 post test. Statistical differences between the percentages of T cells synthesizing cytokines in the PBMC cultures of healthy controls and CML IM patients in response to TCR activation with either SEB or antibody to CD3 were determined by pandora engagement rings the nonpaired t test. P value less than 0.05 was considered statistically significant.Top of pageResultsCytokine synthesis by T cells in whole bloodT cell activation by PMA The ability of T cell subsets in autologous plasma to synthesize IL 2, IFN, TNF, or IL 10 in response to the activation of PKC with PMA was determined in 16 CML IM patients, 19 CML IFN patients, and 20 healthy controls. The percentages of CD4+ and CD8+ T cells that synthesized IL 2, IFN, and TNF after activation with PMA were similar for all groups (Table 1). The percentage of CD4+ T cells but not that of CD8+ T cells that synthesized IL 10 after activation with PMA was higher for CML IFN patients than CML IM patients (P=0.032) and healthy controls (P=0.036). The cultures of whole blood from patients treated with IM had significantly lower percentages of CD4+ T cells that synthesized IL 2 (P=0.017), IFN (P=0.010), and TNF (P=0.009) than those from healthy controls when TCR was activated in the presence of autologous plasma (Table 2). CML IFN patients and healthy controls had similar percentages of SEB activated CD4+ T cells that produced these cytokines. All groups had similar percentages of SEB activated CD8+ T cells that synthesized IL 2, IFN, TNF, and IL 10. These findings indicate that the autologous plasma of CML IM patients may impair cytokine synthesis by activated CD4+ T cells. As the half life of IM is 18 h14 and CML IM patients were on maintenance therapy at the time of this study, we investigated whether cytokine synthesis by activated T cells would be suppressed if the cells were activated in the absence of autologous plasma. In RPMI 1640 medium supplemented with 10% FBS, SEB activated PBMCs of the CML IM patients and control subjects had similar percentages of CD4+ T and CD8+ T cells that synthesized IL 2, IFN, TNF, and IL 10 (Table 3). Under these culture conditions, the CML IM patients and healthy controls had similar percentages of CD4+ and CD8+ T cells that synthesized IL 2, IFN, TNF, and IL 10 (Table 4). These findings indicate that the activation of T cells in the presence of autologous plasma that contained IM may be responsible for the inhibition of cytokine synthesis by the T cells of CML patients on maintenance therapy with IM. Cultures of PBMCs pretreated with exogenous IM followed by activation with an immobilized antibody to CD3 had contained significantly lower percentages of CD4+, but not CD8+, T cells that synthesized IL 2 (P=0.025), IFN (P=0.048), and TNF (P=0.018) than cultures of untreated PBMCs from the same patient (Table 5). Cultures of PBMCs from CML IM patients that were activated in the presence of 10% autologous plasma contained lower percentages of CD4+ T cells that synthesized IL 2, IFN, and TNF than the PBMC cultures that were activated in the presence of 10% FBS (Figure 1a). The suppression of cytokine synthesis was more pronounced in CD4+ T cells than in CD8+ T cells (Figure 1b).Full figure and legend (23K)Cytokine synthesis by anti CD3 activated CD4+ T cells before and after IM therapyTo determine whether cytokine synthesis by CML patients is modified with the initiation of therapy with IM, cytokine synthesis by CD4+ T cells was studied in two CML patients before treatment (at a dose of 400 mg twice a day), and then 5 days later. One of the patients received hydroxyurea as a cytoreductive therapy prior to IM therapy (Figure 2a).

The other patient had received IFN therapy, but it failed (Figure 2b). Before treatment and at 5 days after IM therapy, PBMCs from both patients were suspended in RPMI 1640 medium containing 10% autologous plasma and activated with an immobilized antibody to CD3 and an antibody to CD28. In both cases, the percentages of CD4+ T cells that synthesized IL 2, IFN, and TNF after treatment with IM were lower than those of CD4+ T cells that synthesized these cytokines before therapy with IM.


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