Int J Hematol (2013) 98:558–562 DOI 10.1007/s12185-013-1436-3
ORIGINAL ARTICLE
Clinical significance of Treg cell frequency in acute myeloid leukemia Wenjuan Yang • Yunxiao Xu
Received: 14 February 2013 / Revised: 5 September 2013 / Accepted: 5 September 2013 / Published online: 19 October 2013 Ó The Japanese Society of Hematology 2013
Abstract This study was designed to investigate the clinical significance of peripheral blood CD4? CD25? CD127 low-regulatory T (Treg) cells in acute myeloid leukemia (AML) patients. Treg cells in the peripheral blood of 80 AML patients and 35 age-matched healthy controls were counted by flow cytometry. Correlations between the frequency of circulating Treg cells and disease status, treatment outcome, or prognosis were evaluated. The percentages of Treg cells in patients at diagnosis and during refractory/relapse were significantly higher than that in healthy controls. There was no significant difference in the percentages of Treg cells between patients in remission and healthy controls. After six cycles of chemotherapy, the percentage of Treg cells in patients who achieved complete remission was significantly lower than that in patients at diagnosis, but there was no difference in Treg frequency between refractory/relapse patients and patients at diagnosis. Treg cells in the peripheral blood of AML patients may play a suppressive role in host antitumor immune response. The frequency of Treg cells in peripheral blood may thus be used as a biomarker for predicting sensitivity to chemotherapy and prognosis of AML patients. Additionally, Treg number in peripheral blood could be used to monitor disease status and evaluate disease progression. Keywords Acute myeloid leukemia (AML) Regulatory T cell
W. Yang Y. Xu (&) Department of Hematology, Second Xiangya Hospital, Central South University, 139 Renming Road, Changsha, Hunan 410011, People’s Republic of China e-mail: [email protected]
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Introduction Acute myeloid leukemia (AML) is a malignancy of the myeloid lineage of white blood cells and is characterized by clonal expansion of abnormal myeloid progenitor cells that accumulate in bone marrow and interfere with normal hematopoiesis [1]. Overall, patient survival for AML is rather poor; the 5-year survival rate is 40–50 % in younger adults with AML and drops off dramatically with age [2]. Chemotherapy remains the primary treatment for most AML patients, yet the results are generally unsatisfactory and have shown little improvement for several decades [3]. Given the poor response to chemotherapy alone, immunotherapy is currently being explored as an adjunctive treatment modality for AML. Adoptive cytotoxic T lymphocytes (CTL) therapy has been proposed in cancer therapy for decades; however, clinical trials to date have shown mixed results [4, 5]. Little progress has been made in achieving long-term survival for patients with advanced diseases. Among patients who underwent standard induction chemotherapy, about 20–40 % of patients do not reach complete remission. Even for patients with complete remission, 50–70 % will still relapse after 3 years [6, 7]. However, the mechanism that drives relapse and resistance to remission has not been elucidated. Recently, the adverse role of regulatory T cells in therapeutic efficacy and poor prognosis of AML has drawn more and more attention. Tregs are an immunosuppressive subset of T cells that functions to prevent autoimmunity and regulate physiologic immune reaction [8]. However, Tregs also suppress immune response against cancer [9]. Tregs have been found to exert a profound inhibitory effect on CTL cell-mediated immune response in cancer [10]. Data on the role of Tregs in the progression and prognosis of AML have been accumulating rapidly, but the studies available in literature are
Tregs in acute myeloid leukemia
still small and offer somewhat inconsistent findings. For example, in AML patients, the frequency of Tregs was found to be significantly higher at diagnosis compared with healthy controls [9]. Moreover, high levels of Tregs were observed throughout induction therapy and were found at even greater levels in patients with complete remission (CR) than at diagnosis in some studies [4, 11]. The elevated Tregs level is likely the result of increased proliferation of Treg cells [9] and/or the conversion of CD4-25- T cells into Tregs [12]. Indeed, human AML cells have been found to increase the conversion of CD4-25- T cells into Tregs via modulation of tryptophan catabolism [12]. Multiple mechanisms have been reported to be responsible for the suppressive role of Tregs. For example, Tregs can secrete transforming growth factor-b and IL-10 to exert an inhibitory effect on the immune system [13, 14]. Tregs also inhibit dendritic cell (DC) maturation [15]. Therefore, Tregs depletion can be potentially useful for cancer therapy. However, the efficacy of Treg depletion therapy in AML is controversial [16], which implies that the involvement of Tregs in AML progression and prognosis has not been determined. In this study, we investigated the levels of CD4?CD25?CD127low Treg cells in different disease stages of AML and explored the involvement of Treg cells in disease progression and prognosis. Our study may provide the theoretical basis for the development of AML treatment modalities.
Materials and methods Subjects 80 acute myeloid leukemia patients (42 females and 38 males) with an average age of 43.7 ± 12.2 years (range 23–67 years) were randomly recruited at the Department of Hematology in the Second Xiangya Hospital, Central South University from August 2010 to April 2012. Thirtyfive healthy volunteers who were screened as AML negative (17 females and 18 males) with an average age of 41.9 ± 13.2 years were recruited during the same period of time as controls. All AML patients were diagnosed according to clinical symptoms, bone marrow cytology, immunophenotyping, and cytogenetics (MIC). Among the 80 patients, 6 were M1, 43 were M2 (20 were AML-ETO positive and 23 negative), 11 were M3, 9 were M4, 10 were M5, and 1 was the M6 type. All AML patients were at the acute phase and were treatment-naive. The average counts of peripheral leucocytes were (18.3 ± 5.6) 9 109/L. After recruitment, all patients were treated with DA (daunorubicin: 45 mg/m2, day 1–3; cytarabine: 200 mg/m2, day
559
1–7) or HA (homoharringtonine: 4 mg/m2, day 1–3; cytarabine: 200 mg/m2, day 1–7) regimen to induce remission. After six cycles of chemotherapy (DA, HA, or middle dose Ara-C), 5 patients did not achieve remission, 16 patients relapsed, and 59 patients achieved complete remission. Therefore, of the 80 patients in the diagnosis group, five patients were designated as no remission group, 59 patients were designated as complete remission group, and 16 patients were designated as relapse group. This study complied with the Declaration of Helsinki and was approved by the Ethics Committee of Central South University. Signed informed consent forms were obtained from all subjects who participated in the study. Sample collection Eight milliliter of fasting blood was collected from each subject. Peripheral blood mononuclear cells (PBMC) were separated with lymphocyte separation reagents. Cell morphology was observed, and cell counts were determined under a microscope. CD4? T and CD4? subsets sorting The absolute counts (cells/lL) of mature human helper/ inducer (CD3?CD4?) T lymphocytes and Treg (CD4?CD25?CD127?) cells in erythrocyte lysed whole blood were determined using CD3/CD4 Bitest or CD4/ CD25/CD127 Tritest kit (BD Biosciences, CA, USA) by following the manufacturer’s manual. Briefly, CD3?CD4? cells were sorted by mixing 1 9 106 PBMC cells with 2.5 lg of Jiarufobo acetate (PMA), 1 lg of ionomycin, and 0.7 pg of monensin, followed by incubation with FITCanti-human CD3 monoclonal antibody (mAb) and phycoerythrin (PE)-anti-human CD4 mAb at 37 °C for 5 h in the dark. Afterward, the cells were resuspended in 200 lL of fixative solution, and CD4? T cells were sorted with MoFlo flow cytometry (Dako, CA, USA). To sort Treg cells, each sample of 1 x 106 PBMC cells was incubated with FITC-anti-human CD4 mAb and APCanti-human CD25 mAb as described above. After cells were rinsed with PBS 3 times, 100 lL of fixative solution (4 % polymerisatum) and 1 mL of permeabilization buffer (R&D systems China Co. Ltd., Shanghai, China) were added to the cells, respectively. The cells were then incubated at 37 °C for 1 h in the dark. Next, the cells were then incubated with PE-anti-human CD127 mAb for 2 h at room temperature and rinsed with PBS 3 times. The cells were then resuspended in 200 lL of fixative solution. Treg cells were sorted with MoFlo flow cytometry (Dako, CA, USA) and data were analyzed by FACS Express 3.0 software (De Novo Software, FL, USA).
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Statistical analysis All data were analyzed using SPSS 17.0 software. Results were expressed as mean ± SD. Differences between two groups were analyzed by t test and correlations were analyzed by Spearman rank test. A p \ 0.05 was considered to be statistically significant.
W. Yang, Y. Xu Table 1 Comparison of CD4?CD25?CD127low Treg levels (x±s) Groups
case no
Tregs (%)
At diagnosis
80
22.35 ± 3.08m
Complete remission
59
20.03 ± 1.83
Refractory/relapse
21
24.19 ± 3.13d
Control
35
18.73 ± 2.06
m
p \ 0.05 vs. control and complete remission group
d
p \ 0.05 vs. control and complete remission group
Results General clinical characteristics Among the 80 patients, 6 were M1, 43 were M2, 11 were M3, 9 were M4, 10 were M5, and 1 was M6 type. Among patients with different FAB subtypes, there was no difference in the percentage of Tregs. In patients with M2, no difference in the percentage of Tregs between patients with positive and negative AML-ETO was observed. There were no significant differences in Tregs cell frequency between males and females at different disease stages. Comparison of CD4?CD25?CD127lowTreg frequency in different disease stages The percentages of CD4? CD25?CD127lowTregs in patients at diagnosis and patients with refractory/relapse were significantly higher (p \ 0.05) than in age-matched healthy controls. There was no significant difference in the percentage of CD4? CD25?CD127low Tregs between controls and complete remission patients (p [ 0.05). The percentages of CD4?CD25?CD127low Tregs in patients at diagnosis and patients with refractory/relapse were higher than that in patients that achieved complete remission (p \ 0.05). There was no difference in the percentage of CD4?CD25?CD127low Tregs between patients at diagnosis and patients with refractory/relapse (Table 1; Fig. 1). Our results suggest that Treg frequency can be used to monitor the status of the disease in patients with AML. The effect of chemotherapy on the levels of CD4?CD25?CD127low Tregs Among 59 AML patients with complete remission, the percentage of CD4?CD25?CD127low Tregs cells after 6 cycles of chemotherapy were significantly reduced compared to their percentage at diagnosis (Table 2, p \ 0.05). Among the 21 refractory/relapse patients, the percentage of CD4?CD25?CD127low Tregs showed no difference after 6 cycles of chemotherapy compared to their levels at diagnosis (Table 2, p [ 0.05).
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Fig. 1 The frequencies of Tregs at different disease stages of AML. No difference in Tregs levels was observed between patients at diagnosis, those who acquired complete remission (CR), and patients with refractory/relapse (Ref/Rel) Table 2 The effect of chemotherapy on CD4?CD25?CD127low Treg cell levels (x ± s) AML patients
Before chemotherapy (%)
c After 6 cycles of chemotherapy (%)
59 acquired remission
22.30 ± 3.22
20.03 ± 1.83*
21 refractory/relapse
22.50 ± 2.70
24.19 ± 3.13
*
p \ 0.05 vs. before chemotherapy
Correlation between PB CD4?CD25?CD127low Tregs frequencies and prognosis in patients with AML Among the 80 patients with AML, 5 patients had progressive disease or died within 6 months after disease onset. 59 had acquired complete remission (CR) after 6 months, while 16 patients relapsed after 12 months. Despite the treatments, frequencies of CD4?CD25?CD127low Tregs at diagnosis in PB of patients who had achieved CR were lower than that of patients who had relapsed and had persistent leukemia or died after conventional chemotherapy (Fig. 1). The relapse and progressive disease groups showed significantly higher frequencies of Treg compared to age-matched controls (Fig. 1). To perform correlation analysis between Treg levels and prognosis, a score of 1 was given for patientacquired CR, a score of 2 was given for patient with
Tregs in acute myeloid leukemia
Fig. 2 Correlation analysis between Treg levels and prognosis. Score 1 for patient who acquired complete remission, 2 for patient with relapse, and 3 for dead patient. High prognosis score represents poor prognosis. High Treg levels correlated with poor prognosis in patients with AML
relapse, and a score of 3 was given for dead patients. Our results demonstrated that high Treg frequency significantly correlated with the poor prognosis (Fig. 2) and suggest that Treg frequency could be used to predict clinical outcome in patients with AML.
Discussion The suppressive role of circulating Treg cells on the immune response against cancer has been widely reported. Previous studies of Treg cells in human AML have included patients with untreated disease and patients who achieved complete remission. The correlation between Treg frequency and the disease status or prognosis has been analyzed by several studies with controversial findings. The relationship between Treg cell frequency and outcome of chemotherapy has been rarely analyzed. In this study, we evaluated the frequency of CD4?CD25?CD127low Treg obtained from the peripheral blood of patients with AML at diagnosis and in a subset of these patients who were resistant to induction chemotherapy, achieved CR after induction chemotherapy, or relapsed. The correlation between Treg cell frequency and the disease stages, response to chemotherapy, and prognosis was determined. In comparison to age-matched healthy controls, newly diagnosed AML patients had an increased frequency of Treg in their peripheral blood. An elevated frequency of Treg in the peripheral blood has been previously reported in various solid cancers [17] and AML patients [9]. Increases of Treg number at the tumor site or in the peripheral blood of cancer patients have been previously associated with poor treatment outcomes of these diseases [18, 19]. In this study, we found that the percentage of CD4?CD25?CD127low Tregs in
561
AML patients with complete remission was significantly reduced after 6 cycles of chemotherapy compared to the percentage before therapy. In contrast, in refractory/relapse patients, the percentage of Tregs showed no significant changes after 6 cycles of chemotherapy when compared with the percentage of Tregs at diagnosis. We further analyzed the percentage of Tregs at diagnosis and its association with disease status. We found that the patients who relapsed and developed progressive disease after chemotherapy had significantly higher frequencies of Treg compared to agematched controls. Our results suggest that patients with high Treg percentage in the periphery blood have low sensitivity to chemotherapy, and the level of Treg cells in the periphery blood could be used as a cellular marker to predict a patient’s response to chemotherapy. However, reports on the frequencies of Treg in complete remission patients are controversial. Zhang et al. [20] demonstrated that CD4?CD25?CD127low Treg frequencies were reduced when patients achieved complete remission. Szczepanski et al. [4] revealed that CD4?CD25highFoxp3? Treg frequency remained elevated in patients who achieved CR after induction therapy. The authors hypothesized that the high frequency of Treg post-therapy may represent a secondary response to inflammation caused by induction chemotherapy and cytokine secretion, which promote the expansion and proliferation of peripheral Treg. However, this hypothesis is unable to explain the suppressive role of Treg in tumor immunity observed in most studies. The inhibitory role of chemotherapy on Treg frequency and activity has been previously observed in both animal models and clinical settings [21, 22]. However, the mechanism responsible for selective reduction of Treg cells after chemotherapy is currently unclear. A previous study observed a re-elevation of Treg cells in the peripheral blood of relapsed AML patients [20]. In this study, the percentage of CD4? CD25?CD127lowTregs in the peripheral blood of AML patients who relapsed was significantly higher than in patients who achieved complete remission. The percentage of CD4?CD25?CD127low Tregs in relapsed patients was re-elevated to the level of patients at diagnosis. This may suggest that Treg frequency could be used to monitor the status of the disease in patients with AML. In addition, we found that the frequency of CD4?CD25?CD127low Tregs at diagnosis in PB of patients who had achieved complete remission was lower than that in PB of patients who relapsed and had persistent leukemia or died after conventional chemotherapy. This suggests that Treg frequency at diagnosis could be used to predict the clinical outcome in patients with AML. In summary, our data provide insights into the clinical significance of Tregs in patients with AML. The increased frequency of Tregs in AML patients at diagnosis and decreased frequency after achieving complete remission
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W. Yang, Y. Xu
indicate that these cells may play a role in host antitumor immune responses. Our data suggest that the frequency of Treg cells in peripheral blood at diagnosis could be used as a cellular marker for predicting a patient’s sensitivity to chemotherapy and disease prognosis of AML patients. Our data also suggest that Tregs frequency in peripheral blood could be used to monitor disease status and evaluate disease progression. Although further studies are required to confirm these hypotheses, our study suggests that depletion of Treg should be considered a viable therapy for patients with AML. Conflict of interest
All authors declare no conflict of interest.
References 1. Smits EL, Berneman ZN, Van Tendeloo VF. Immunotherapy of acute myeloid leukemia: current approaches. Oncologist. 2009;14:240–52. 2. Kanakry CG, Hess AD, Gocke CD, Thoburn C, Kos F, Meyer C, et al. Early lymphocyte recovery after intensive timed sequential chemotherapy for acute myelogenous leukemia: peripheral oligo clonal expansion of regulatory T cells. Blood. 2011;117:608–17. 3. Ustun C, Miller JS, Munn DH, Weisdorf DJ, Blazar BR. Regulatory T cells in acute myelogenous leukemia: is it time for immunomodulation? Blood. 2011;118:5084–95. 4. Szczepanski MJ, Szajnik M, Czystowska M, Mandapathil M, Strauss L, Welsh A, et al. Increased frequency and suppression by regulatory T cells in patients with acute myelogenous leukemia. Clin Cancer Res. 2009;15:3325–32. 5. Pandolfi F, Cianci R, Lolli S, Dunn IS, Newton EE, Haggerty TJ, et al. Strategies to overcome obstacles to successful immunotherapy of melanoma. Int J Immunopathol Pharmacol. 2008;21: 493–500. 6. Robak T, Wrzesie0 n-Ku0 s A. The search for optimal treatment in relapsed and refractory acute myeloid leukemia. Leuk Lymphoma. 2002;43:281–91. 7. Mato AR, Morgans A, Luger SM. Novel strategies for relapsed and refractory acute myeloid leukemia. Curr Opin Hematol. 2008;15:108–14. 8. Kelley TW, Parker CJ. CD4(?)CD25(?)Foxp3(?) regulatory T cells and hematologic malignancies. Front Biosci (Schol Ed). 2010;2:980–92. 9. Wang X, Zheng J, Liu J, Yao J, He Y, Li X, et al. Increased population of CD4(?)CD25(high), regulatory T cells with their higher apoptotic and proliferating status in peripheral blood of acute myeloid leukemia patients. Eur J Haematol. 2005;75: 468–76.
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10. Fu J, Xu D, Liu Z, Shi M, Zhao P, Fu B, et al. Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients. Gastroenterology. 2007;132:2328–39. 11. Ersvaer E, Liseth K, Skavland J, Gjertsen BT, Bruserud O. Intensive chemotherapy for acute myeloid leukemia differentially affects circulating TC1, TH1, TH17 and TREG cells. BMC Immunol. 2010;11:38. 12. Curti A, Pandolfi S, Valzasina B, Aluigi M, Isidori A, Ferri E, et al. Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25- into CD25? T regulatory cells. Blood. 2007;109:2871–7. 13. Larmonier N, Marron M, Zeng Y, Cantrell J, Romanoski A, Sepassi M, et al. Tumor derived CD4-CD25- regulatory T cell suppression of dendritic cell function involves TGFbeta and IL10. Cancer Immunol Immunother. 2007;56:48–59. 14. Clark RA, Huang SJ, Murphy GF, Mollet IG, Hijnen D, Muthukuru M, et al. Human squamous cell carcinomas evade the immune response by down-regulation of vascular E-selectin and recruitment of regulatory T cells. J Exp Med. 2008;205:2221–34. 15. Grauer OM, Sutmuller RP, van Maren W, Jacobs JF, Bennink E, Toonen LW, et al. Elimination of regulatory T cells is essential for an effective vaccination with tumor lysate-pulsed dendritic cells in a murine glioma model. Int J Cancer. 2008;122: 1794–802. 16. Ustun C, Miller JS, Munn DH, Weisdorf DJ, Blazar BR. Regulatory T cells in acute myelogenous leukemia: is it time for immunomodulation? Blood. 2011;118:5084–95. 17. Beyer M, Schultze JL. Regulatory T cells in cancer. Blood. 2006;108:804–11. 18. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med. 2004;10:942–9. 19. Griffiths RW, Elkord E, Gilham DE, Ramani V, Clarke N, Stern PL, et al. Frequency of regulatory T cells in renal cell carcinoma patients and investigation of correlation with survival. Cancer Immunol Immunother. 2007;56:1743–53. 20. Zhang S, Han Y, Wu J, Yu K, Bi L, Zhuang Y, et al. Elevated frequencies of CD4? CD25? CD127lo regulatory T cells is associated to poor prognosis in patients with acute myeloid leukemia. Int J Cancer. 2011;129:1373–81. 21. Ghiringhelli F, Menard C, Puig PE, Ladoire S, Roux S, Martin F, et al. Metronomic cyclophosphamide regimen selectively depletes CD4?CD25?regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer Immunol Immunother. 2007;56:641–8. 22. Chu Y, Wang LX, Yang G, Ross HJ, Urba WJ, Prell R, et al. Efficacy of GM-CSF-producing tumor vaccine after docetaxel chemotherapy in mice bearing established Lewis lung carcinoma. J Immunother. 2006;29:367–80.
ORIGINAL ARTICLE
Clinical significance of Treg cell frequency in acute myeloid leukemia Wenjuan Yang • Yunxiao Xu
Received: 14 February 2013 / Revised: 5 September 2013 / Accepted: 5 September 2013 / Published online: 19 October 2013 Ó The Japanese Society of Hematology 2013
Abstract This study was designed to investigate the clinical significance of peripheral blood CD4? CD25? CD127 low-regulatory T (Treg) cells in acute myeloid leukemia (AML) patients. Treg cells in the peripheral blood of 80 AML patients and 35 age-matched healthy controls were counted by flow cytometry. Correlations between the frequency of circulating Treg cells and disease status, treatment outcome, or prognosis were evaluated. The percentages of Treg cells in patients at diagnosis and during refractory/relapse were significantly higher than that in healthy controls. There was no significant difference in the percentages of Treg cells between patients in remission and healthy controls. After six cycles of chemotherapy, the percentage of Treg cells in patients who achieved complete remission was significantly lower than that in patients at diagnosis, but there was no difference in Treg frequency between refractory/relapse patients and patients at diagnosis. Treg cells in the peripheral blood of AML patients may play a suppressive role in host antitumor immune response. The frequency of Treg cells in peripheral blood may thus be used as a biomarker for predicting sensitivity to chemotherapy and prognosis of AML patients. Additionally, Treg number in peripheral blood could be used to monitor disease status and evaluate disease progression. Keywords Acute myeloid leukemia (AML) Regulatory T cell
W. Yang Y. Xu (&) Department of Hematology, Second Xiangya Hospital, Central South University, 139 Renming Road, Changsha, Hunan 410011, People’s Republic of China e-mail: [email protected]
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Introduction Acute myeloid leukemia (AML) is a malignancy of the myeloid lineage of white blood cells and is characterized by clonal expansion of abnormal myeloid progenitor cells that accumulate in bone marrow and interfere with normal hematopoiesis [1]. Overall, patient survival for AML is rather poor; the 5-year survival rate is 40–50 % in younger adults with AML and drops off dramatically with age [2]. Chemotherapy remains the primary treatment for most AML patients, yet the results are generally unsatisfactory and have shown little improvement for several decades [3]. Given the poor response to chemotherapy alone, immunotherapy is currently being explored as an adjunctive treatment modality for AML. Adoptive cytotoxic T lymphocytes (CTL) therapy has been proposed in cancer therapy for decades; however, clinical trials to date have shown mixed results [4, 5]. Little progress has been made in achieving long-term survival for patients with advanced diseases. Among patients who underwent standard induction chemotherapy, about 20–40 % of patients do not reach complete remission. Even for patients with complete remission, 50–70 % will still relapse after 3 years [6, 7]. However, the mechanism that drives relapse and resistance to remission has not been elucidated. Recently, the adverse role of regulatory T cells in therapeutic efficacy and poor prognosis of AML has drawn more and more attention. Tregs are an immunosuppressive subset of T cells that functions to prevent autoimmunity and regulate physiologic immune reaction [8]. However, Tregs also suppress immune response against cancer [9]. Tregs have been found to exert a profound inhibitory effect on CTL cell-mediated immune response in cancer [10]. Data on the role of Tregs in the progression and prognosis of AML have been accumulating rapidly, but the studies available in literature are
Tregs in acute myeloid leukemia
still small and offer somewhat inconsistent findings. For example, in AML patients, the frequency of Tregs was found to be significantly higher at diagnosis compared with healthy controls [9]. Moreover, high levels of Tregs were observed throughout induction therapy and were found at even greater levels in patients with complete remission (CR) than at diagnosis in some studies [4, 11]. The elevated Tregs level is likely the result of increased proliferation of Treg cells [9] and/or the conversion of CD4-25- T cells into Tregs [12]. Indeed, human AML cells have been found to increase the conversion of CD4-25- T cells into Tregs via modulation of tryptophan catabolism [12]. Multiple mechanisms have been reported to be responsible for the suppressive role of Tregs. For example, Tregs can secrete transforming growth factor-b and IL-10 to exert an inhibitory effect on the immune system [13, 14]. Tregs also inhibit dendritic cell (DC) maturation [15]. Therefore, Tregs depletion can be potentially useful for cancer therapy. However, the efficacy of Treg depletion therapy in AML is controversial [16], which implies that the involvement of Tregs in AML progression and prognosis has not been determined. In this study, we investigated the levels of CD4?CD25?CD127low Treg cells in different disease stages of AML and explored the involvement of Treg cells in disease progression and prognosis. Our study may provide the theoretical basis for the development of AML treatment modalities.
Materials and methods Subjects 80 acute myeloid leukemia patients (42 females and 38 males) with an average age of 43.7 ± 12.2 years (range 23–67 years) were randomly recruited at the Department of Hematology in the Second Xiangya Hospital, Central South University from August 2010 to April 2012. Thirtyfive healthy volunteers who were screened as AML negative (17 females and 18 males) with an average age of 41.9 ± 13.2 years were recruited during the same period of time as controls. All AML patients were diagnosed according to clinical symptoms, bone marrow cytology, immunophenotyping, and cytogenetics (MIC). Among the 80 patients, 6 were M1, 43 were M2 (20 were AML-ETO positive and 23 negative), 11 were M3, 9 were M4, 10 were M5, and 1 was the M6 type. All AML patients were at the acute phase and were treatment-naive. The average counts of peripheral leucocytes were (18.3 ± 5.6) 9 109/L. After recruitment, all patients were treated with DA (daunorubicin: 45 mg/m2, day 1–3; cytarabine: 200 mg/m2, day
559
1–7) or HA (homoharringtonine: 4 mg/m2, day 1–3; cytarabine: 200 mg/m2, day 1–7) regimen to induce remission. After six cycles of chemotherapy (DA, HA, or middle dose Ara-C), 5 patients did not achieve remission, 16 patients relapsed, and 59 patients achieved complete remission. Therefore, of the 80 patients in the diagnosis group, five patients were designated as no remission group, 59 patients were designated as complete remission group, and 16 patients were designated as relapse group. This study complied with the Declaration of Helsinki and was approved by the Ethics Committee of Central South University. Signed informed consent forms were obtained from all subjects who participated in the study. Sample collection Eight milliliter of fasting blood was collected from each subject. Peripheral blood mononuclear cells (PBMC) were separated with lymphocyte separation reagents. Cell morphology was observed, and cell counts were determined under a microscope. CD4? T and CD4? subsets sorting The absolute counts (cells/lL) of mature human helper/ inducer (CD3?CD4?) T lymphocytes and Treg (CD4?CD25?CD127?) cells in erythrocyte lysed whole blood were determined using CD3/CD4 Bitest or CD4/ CD25/CD127 Tritest kit (BD Biosciences, CA, USA) by following the manufacturer’s manual. Briefly, CD3?CD4? cells were sorted by mixing 1 9 106 PBMC cells with 2.5 lg of Jiarufobo acetate (PMA), 1 lg of ionomycin, and 0.7 pg of monensin, followed by incubation with FITCanti-human CD3 monoclonal antibody (mAb) and phycoerythrin (PE)-anti-human CD4 mAb at 37 °C for 5 h in the dark. Afterward, the cells were resuspended in 200 lL of fixative solution, and CD4? T cells were sorted with MoFlo flow cytometry (Dako, CA, USA). To sort Treg cells, each sample of 1 x 106 PBMC cells was incubated with FITC-anti-human CD4 mAb and APCanti-human CD25 mAb as described above. After cells were rinsed with PBS 3 times, 100 lL of fixative solution (4 % polymerisatum) and 1 mL of permeabilization buffer (R&D systems China Co. Ltd., Shanghai, China) were added to the cells, respectively. The cells were then incubated at 37 °C for 1 h in the dark. Next, the cells were then incubated with PE-anti-human CD127 mAb for 2 h at room temperature and rinsed with PBS 3 times. The cells were then resuspended in 200 lL of fixative solution. Treg cells were sorted with MoFlo flow cytometry (Dako, CA, USA) and data were analyzed by FACS Express 3.0 software (De Novo Software, FL, USA).
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560
Statistical analysis All data were analyzed using SPSS 17.0 software. Results were expressed as mean ± SD. Differences between two groups were analyzed by t test and correlations were analyzed by Spearman rank test. A p \ 0.05 was considered to be statistically significant.
W. Yang, Y. Xu Table 1 Comparison of CD4?CD25?CD127low Treg levels (x±s) Groups
case no
Tregs (%)
At diagnosis
80
22.35 ± 3.08m
Complete remission
59
20.03 ± 1.83
Refractory/relapse
21
24.19 ± 3.13d
Control
35
18.73 ± 2.06
m
p \ 0.05 vs. control and complete remission group
d
p \ 0.05 vs. control and complete remission group
Results General clinical characteristics Among the 80 patients, 6 were M1, 43 were M2, 11 were M3, 9 were M4, 10 were M5, and 1 was M6 type. Among patients with different FAB subtypes, there was no difference in the percentage of Tregs. In patients with M2, no difference in the percentage of Tregs between patients with positive and negative AML-ETO was observed. There were no significant differences in Tregs cell frequency between males and females at different disease stages. Comparison of CD4?CD25?CD127lowTreg frequency in different disease stages The percentages of CD4? CD25?CD127lowTregs in patients at diagnosis and patients with refractory/relapse were significantly higher (p \ 0.05) than in age-matched healthy controls. There was no significant difference in the percentage of CD4? CD25?CD127low Tregs between controls and complete remission patients (p [ 0.05). The percentages of CD4?CD25?CD127low Tregs in patients at diagnosis and patients with refractory/relapse were higher than that in patients that achieved complete remission (p \ 0.05). There was no difference in the percentage of CD4?CD25?CD127low Tregs between patients at diagnosis and patients with refractory/relapse (Table 1; Fig. 1). Our results suggest that Treg frequency can be used to monitor the status of the disease in patients with AML. The effect of chemotherapy on the levels of CD4?CD25?CD127low Tregs Among 59 AML patients with complete remission, the percentage of CD4?CD25?CD127low Tregs cells after 6 cycles of chemotherapy were significantly reduced compared to their percentage at diagnosis (Table 2, p \ 0.05). Among the 21 refractory/relapse patients, the percentage of CD4?CD25?CD127low Tregs showed no difference after 6 cycles of chemotherapy compared to their levels at diagnosis (Table 2, p [ 0.05).
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Fig. 1 The frequencies of Tregs at different disease stages of AML. No difference in Tregs levels was observed between patients at diagnosis, those who acquired complete remission (CR), and patients with refractory/relapse (Ref/Rel) Table 2 The effect of chemotherapy on CD4?CD25?CD127low Treg cell levels (x ± s) AML patients
Before chemotherapy (%)
c After 6 cycles of chemotherapy (%)
59 acquired remission
22.30 ± 3.22
20.03 ± 1.83*
21 refractory/relapse
22.50 ± 2.70
24.19 ± 3.13
*
p \ 0.05 vs. before chemotherapy
Correlation between PB CD4?CD25?CD127low Tregs frequencies and prognosis in patients with AML Among the 80 patients with AML, 5 patients had progressive disease or died within 6 months after disease onset. 59 had acquired complete remission (CR) after 6 months, while 16 patients relapsed after 12 months. Despite the treatments, frequencies of CD4?CD25?CD127low Tregs at diagnosis in PB of patients who had achieved CR were lower than that of patients who had relapsed and had persistent leukemia or died after conventional chemotherapy (Fig. 1). The relapse and progressive disease groups showed significantly higher frequencies of Treg compared to age-matched controls (Fig. 1). To perform correlation analysis between Treg levels and prognosis, a score of 1 was given for patientacquired CR, a score of 2 was given for patient with
Tregs in acute myeloid leukemia
Fig. 2 Correlation analysis between Treg levels and prognosis. Score 1 for patient who acquired complete remission, 2 for patient with relapse, and 3 for dead patient. High prognosis score represents poor prognosis. High Treg levels correlated with poor prognosis in patients with AML
relapse, and a score of 3 was given for dead patients. Our results demonstrated that high Treg frequency significantly correlated with the poor prognosis (Fig. 2) and suggest that Treg frequency could be used to predict clinical outcome in patients with AML.
Discussion The suppressive role of circulating Treg cells on the immune response against cancer has been widely reported. Previous studies of Treg cells in human AML have included patients with untreated disease and patients who achieved complete remission. The correlation between Treg frequency and the disease status or prognosis has been analyzed by several studies with controversial findings. The relationship between Treg cell frequency and outcome of chemotherapy has been rarely analyzed. In this study, we evaluated the frequency of CD4?CD25?CD127low Treg obtained from the peripheral blood of patients with AML at diagnosis and in a subset of these patients who were resistant to induction chemotherapy, achieved CR after induction chemotherapy, or relapsed. The correlation between Treg cell frequency and the disease stages, response to chemotherapy, and prognosis was determined. In comparison to age-matched healthy controls, newly diagnosed AML patients had an increased frequency of Treg in their peripheral blood. An elevated frequency of Treg in the peripheral blood has been previously reported in various solid cancers [17] and AML patients [9]. Increases of Treg number at the tumor site or in the peripheral blood of cancer patients have been previously associated with poor treatment outcomes of these diseases [18, 19]. In this study, we found that the percentage of CD4?CD25?CD127low Tregs in
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AML patients with complete remission was significantly reduced after 6 cycles of chemotherapy compared to the percentage before therapy. In contrast, in refractory/relapse patients, the percentage of Tregs showed no significant changes after 6 cycles of chemotherapy when compared with the percentage of Tregs at diagnosis. We further analyzed the percentage of Tregs at diagnosis and its association with disease status. We found that the patients who relapsed and developed progressive disease after chemotherapy had significantly higher frequencies of Treg compared to agematched controls. Our results suggest that patients with high Treg percentage in the periphery blood have low sensitivity to chemotherapy, and the level of Treg cells in the periphery blood could be used as a cellular marker to predict a patient’s response to chemotherapy. However, reports on the frequencies of Treg in complete remission patients are controversial. Zhang et al. [20] demonstrated that CD4?CD25?CD127low Treg frequencies were reduced when patients achieved complete remission. Szczepanski et al. [4] revealed that CD4?CD25highFoxp3? Treg frequency remained elevated in patients who achieved CR after induction therapy. The authors hypothesized that the high frequency of Treg post-therapy may represent a secondary response to inflammation caused by induction chemotherapy and cytokine secretion, which promote the expansion and proliferation of peripheral Treg. However, this hypothesis is unable to explain the suppressive role of Treg in tumor immunity observed in most studies. The inhibitory role of chemotherapy on Treg frequency and activity has been previously observed in both animal models and clinical settings [21, 22]. However, the mechanism responsible for selective reduction of Treg cells after chemotherapy is currently unclear. A previous study observed a re-elevation of Treg cells in the peripheral blood of relapsed AML patients [20]. In this study, the percentage of CD4? CD25?CD127lowTregs in the peripheral blood of AML patients who relapsed was significantly higher than in patients who achieved complete remission. The percentage of CD4?CD25?CD127low Tregs in relapsed patients was re-elevated to the level of patients at diagnosis. This may suggest that Treg frequency could be used to monitor the status of the disease in patients with AML. In addition, we found that the frequency of CD4?CD25?CD127low Tregs at diagnosis in PB of patients who had achieved complete remission was lower than that in PB of patients who relapsed and had persistent leukemia or died after conventional chemotherapy. This suggests that Treg frequency at diagnosis could be used to predict the clinical outcome in patients with AML. In summary, our data provide insights into the clinical significance of Tregs in patients with AML. The increased frequency of Tregs in AML patients at diagnosis and decreased frequency after achieving complete remission
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indicate that these cells may play a role in host antitumor immune responses. Our data suggest that the frequency of Treg cells in peripheral blood at diagnosis could be used as a cellular marker for predicting a patient’s sensitivity to chemotherapy and disease prognosis of AML patients. Our data also suggest that Tregs frequency in peripheral blood could be used to monitor disease status and evaluate disease progression. Although further studies are required to confirm these hypotheses, our study suggests that depletion of Treg should be considered a viable therapy for patients with AML. Conflict of interest
All authors declare no conflict of interest.
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