International Journal of Hematology

, Volume 108, Issue 6, pp 588–597 | Cite as

Dasatinib-induced anti-leukemia cellular immunity through a novel subset of CD57 positive helper/cytotoxic CD4 T cells in chronic myelogenous leukemia patients

  • Naoki Watanabe
  • Tomoiku TakakuEmail author
  • Kazuyoshi Takeda
  • Shuichi Shirane
  • Tokuko Toyota
  • Michiaki Koike
  • Masaaki Noguchi
  • Takao Hirano
  • Hiroshi Fujiwara
  • Norio Komatsu
Original Article


Dasatinib induces lymphocytosis of large granular lymphocytes (LGLs) in a proportion of patients with chronic myelogenous leukemia (CML), and is associated with better clinical outcomes. LGLs consist of cytotoxic T lymphocytes and natural killer cells; however, the context and phenotypic/functional features of each type of LGL are unknown. To better define features of these LGLs, we investigated lymphocytosis in CML patients treated with dasatinib. D57-positive and CD4-positive type I T-helper (Th) cells (CD57+ Th cells) rarely occur in CML patients without lymphocytosis and in healthy individuals; however, a substantial increase in the proportion of CD57+ Th cells was observed in CML patients treated with dasatinib. In addition, these cells showed appreciable levels of cytocidal activity via cytotoxic degranulation. Analysis of T-cell receptor α and β sequences showed a skewed T-cell repertoire in the CD57+ Th cells. Furthermore, patients with LGLs and CD57+ Th lymphocytosis achieved stronger molecular responses than did those without lymphocytosis. While further studies are warranted, our observations suggest that dasatinib induces the expansion of CD57+ Th-LGLs, which may play a crucial role in the dasatinib-induced response against Philadelphia chromosome-positive leukemia.


CD57 expression Chronic myelogenous leukemia Cytotoxic CD4+ T cell Dasatinib Large granular lymphocyte 



We are grateful to Ryuji Suzuki and Kazutaka Kitaura from Repertoire Genesis Incorporated, for providing technical support.

Author contributions

NW performed the study, analyzed the results, and wrote the paper; TTa designed the research, analyzed the results, wrote the paper, and directed the research; KT and SS analyzed the results; TTo performed the study; MK, MN, and TH contributed analytical materials; HF discussed and analyzed data, provided experimental concepts and materials, and edited the paper; and NK directed the research.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest. A summary of relevant information will be published with the manuscript.

Supplementary material

12185_2018_2517_MOESM1_ESM.tif (24.9 mb)
Supplemental Figure 1 Representative immunophenotyping analysis of large granular lymphocytes (LGLs). CD56+ natural killer (NK) cells in patients (a) without and (b) with LGL lymphocytosis. CD57+ NK cells in patients (c) without and with (d) LGL lymphocytosis. CD57+ cytotoxic T lymphocytes in patients (e) without and (f) with LGL lymphocytosis. CD57+ helper T cells in patients (g) without and (h) with LGL lymphocytosis (TIF 25509 KB)
12185_2018_2517_MOESM2_ESM.tif (24.9 mb)
Supplemental Figure 2 Comparison of cytoplasmic granules in CD57-positive and -negative Th cells. Cytoplasmic granules by Giemsa staining indicated morphologic features of sorted CD57 positive and negative helper T (Th) cells. (a) CD57+ Th cells showed cytoplasmic granules. (b) CD57 negative Th cells did not show cytoplasmic granules. (TIF 25507 KB)
12185_2018_2517_MOESM3_ESM.tif (27.2 mb)
Supplemental Figure 3 Immunophenotyping analysis of CD57+ Th cells. Flow cytometric analysis of representative T cells. (a) Expression of CD45RA and CCR7. (b) interferon (IFN)-γ and interleukin (IL)-4. (c) IFN-γ and IL-17 in CD57+ Th cells. T-cell receptor (TCR) Vα24-Jα18 expression in (d) CD57+ Th cells, (e) CD3+ CD4- CD57+ cells, and in (f) CD3+ CD4− CD57− cells. (TIF 27896 KB)
12185_2018_2517_MOESM4_ESM.tif (24.9 mb)
Supplemental Figure 4 Analysis of intracellular cytokine production without stimulation in CD57+ helper T (Th) cells. CD57+ Th cells without phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) stimulation did not produce any cytokine. (a) interferon (IFN)-γ and interleukin (IL)-4. (b) IFN-γ and IL-17. For intracellular cytokine staining, the sorted effector cells were cultured with PMA (50 ng/mL) and ION (500 ng/mL) (Sigma-Aldrich) for 12 h, with the addition of Brefeldin A (Sigma-Aldrich) 2 h before the end of incubation. The cells were then fixed with 4% paraformaldehyde, permeabilized with permeabilization buffer (eBioscience), and stained with antibodies. (TIF 25508 KB)
12185_2018_2517_MOESM5_ESM.tif (24.9 mb)
Supplemental Figure 5 Cytotoxicity of CD57+/− helper T (Th) cells targeting K562-A24/C II TA and K562-A24. The solid line represents the cytotoxicity of CD3+ CD4+ CD57+ Th cells and the broken line represents the cytotoxicity of CD3+ CD4+ CD57− Th cells. (TIF 25507 KB)


  1. 1.
    Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley JF, et al. European LeukemiaNet recommendations for the management of chronic myeloid leukemia: 2013. Blood. 2013;122:872–84.CrossRefGoogle Scholar
  2. 2.
    Hochhaus A, Larson RA, Guilhot F, Radich JP, Branford S, Hughes TP, et al. Long-term outcomes of imatinib treatment for chronic myeloid leukemia. N Engl J Med. 2017;376:917–27.CrossRefGoogle Scholar
  3. 3.
    Steinberg M. Dasatinib: a tyrosine kinase inhibitor for the treatment of chronic myelogenous leukemia and philadelphia chromosome-positive acute lymphoblastic leukemia. Clin Ther. 2007;29:2289–308.CrossRefGoogle Scholar
  4. 4.
    Hochhaus A, Saglio G, Hughes TP, Larson RA, Kim DW, Issaragrisil S, et al. Long-term benefits and risks of frontline nilotinib vs imatinib for chronic myeloid leukemia in chronic phase: 5-year update of the randomized ENESTnd trial. Leukemia. 2016;30:1044–54.CrossRefGoogle Scholar
  5. 5.
    Cortes JE, Saglio G, Kantarjian HM, Baccarani M, Mayer J, Boque C, et al. Final 5-year study results of DASISION: the dasatinib versus imatinib study in treatment-naive chronic myeloid leukemia patients trial. J Clin Oncol. 2016;34:2333–40.CrossRefGoogle Scholar
  6. 6.
    Nakamae H, Fujisawa S, Ogura M, Uchida T, Onishi Y, Taniwaki M, et al. Dasatinib versus imatinib in Japanese patients with newly diagnosed chronic phase chronic myeloid leukemia: a subanalysis of the DASISION 5-year final report. Int J Hematol. 2017;105:792–804.CrossRefGoogle Scholar
  7. 7.
    Nakamae H, Fukuda T, Nakaseko C, Kanda Y, Ohmine K, Ono T, et al. Nilotinib vs. imatinib in Japanese patients with newly diagnosed chronic myeloid leukemia in chronic phase: long-term follow-up of the Japanese subgroup of the randomized ENESTnd trial. Int J Hematol. 2018;107:327–36.CrossRefGoogle Scholar
  8. 8.
    Takaku T, Iriyama N, Mitsumori T, Sato E, Gotoh A, Kirito K, et al. Clinical efficacy and safety of first-line dasatinib therapy and the relevance of velocity of BCR-ABL1 transcript decline for achievement of molecular responses in newly diagnosed chronic-phase chronic myeloid leukemia: report from the Juntendo Yamanashi Cooperative Study Group. Oncology. 2018;94:85–91.CrossRefGoogle Scholar
  9. 9.
    Steegmann JL, Baccarani M, Breccia M, Casado LF, Garcia-Gutierrez V, Hochhaus A, et al. European LeukemiaNet recommendations for the management and avoidance of adverse events of treatment in chronic myeloid leukaemia. Leukemia. 2016;30:1648–71.CrossRefGoogle Scholar
  10. 10.
    Kim DH, Kamel-Reid S, Chang H, Sutherland R, Jung CW, Kim HJ, et al. Natural killer or natural killer/T cell lineage large granular lymphocytosis associated with dasatinib therapy for Philadelphia chromosome positive leukemia. Haematologica. 2009;94:135–9.CrossRefGoogle Scholar
  11. 11.
    Mustjoki S, Ekblom M, Arstila TP, Dybedal I, Epling-Burnette PK, Guilhot F, et al. Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy. Leukemia. 2009;23:1398–405.CrossRefGoogle Scholar
  12. 12.
    Kreutzman A, Juvonen V, Kairisto V, Ekblom M, Stenke L, Seggewiss R, et al. Mono/oligoclonal T and NK cells are common in chronic myeloid leukemia patients at diagnosis and expand during dasatinib therapy. Blood. 2010;116:772–82.CrossRefGoogle Scholar
  13. 13.
    Kumagai T, Matsuki E, Inokuchi K, Ohashi K, Shinagawa A, Takeuchi J, et al. Relative increase in lymphocytes from as early as 1 month predicts improved response to dasatinib in chronic-phase chronic myelogenous leukemia. Int J Hematol. 2014;99:41–52.CrossRefGoogle Scholar
  14. 14.
    Nagata Y, Ohashi K, Fukuda S, Kamata N, Akiyama H, Sakamaki H. Clinical features of dasatinib-induced large granular lymphocytosis and pleural effusion. Int J Hematol. 2010;91:799–807.CrossRefGoogle Scholar
  15. 15.
    Hantschel O, Rix U, Schmidt U, Burckstummer T, Kneidinger M, Schutze G, et al. The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib. Proc Natl Acad Sci U S A. 2007;104:13283–8.CrossRefGoogle Scholar
  16. 16.
    Rix U, Hantschel O, Durnberger G, Remsing Rix LL, Planyavsky M, Fernbach NV, et al. Chemical proteomic profiles of the BCR-ABL inhibitors imatinib, nilotinib, and dasatinib reveal novel kinase and nonkinase targets. Blood. 2007;110:4055–63.CrossRefGoogle Scholar
  17. 17.
    Bantscheff M, Eberhard D, Abraham Y, Bastuck S, Boesche M, Hobson S, et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat Biotechnol. 2007;25:1035–44.CrossRefGoogle Scholar
  18. 18.
    Sallusto F, Lanzavecchia A. Exploring pathways for memory T cell generation. J Clin Investig. 2001;108:805–6.CrossRefGoogle Scholar
  19. 19.
    Friberg DD, Bryant JL, Whiteside TL. Measurements of natural killer (NK) activity and NK-cell quantification. Methods. 1996;9:316–26.CrossRefGoogle Scholar
  20. 20.
    Fujiwara H, Ochi T, Ochi F, Miyazaki Y, Asai H, Narita M, et al. Antileukemia multifunctionality of CD4(+) T cells genetically engineered by HLA class I-restricted and WT1-specific T-cell receptor gene transfer. Leukemia. 2015;29:2393–401.CrossRefGoogle Scholar
  21. 21.
    Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 2013;48:452–8.CrossRefGoogle Scholar
  22. 22.
    Palmer BE, Blyveis N, Fontenot AP, Wilson CC. Functional and phenotypic characterization of CD57+ CD4+ T cells and their association with HIV-1-induced T cell dysfunction. J Immunol. 2005;175:8415–23.CrossRefGoogle Scholar
  23. 23.
    Bjorkstrom NK, Riese P, Heuts F, Andersson S, Fauriat C, Ivarsson MA, et al. Expression patterns of NKG2A, KIR, and CD57 define a process of CD56dim NK-cell differentiation uncoupled from NK-cell education. Blood. 2010;116:3853–64.CrossRefGoogle Scholar
  24. 24.
    Brenchley JM, Karandikar NJ, Betts MR, Ambrozak DR, Hill BJ, Crotty LE, et al. Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T cells. Blood. 2003;101:2711–20.CrossRefGoogle Scholar
  25. 25.
    Legac E, Autran B, Merle-Beral H, Katlama C, Debre P. CD4+ CD7− CD57+ T cells: a new T-lymphocyte subset expanded during human immunodeficiency virus infection. Blood. 1992;79:1746–53.PubMedGoogle Scholar
  26. 26.
    Watanabe H, Weerasinghe A, Miyaji C, Sekikawa H, Toyabe S, Mannor MK, et al. Expansion of unconventional T cells with natural killer markers in malaria patients. Parasitol Int. 2003;52:61–70.CrossRefGoogle Scholar
  27. 27.
    Jimenez-Martinez MC, Linares M, Baez R, Montano LF, Martinez-Cairo S, Gorocica P, et al. Intracellular expression of interleukin-4 and interferon-gamma by a Mycobacterium tuberculosis antigen-stimulated CD4+ CD57+ T-cell subpopulation with memory phenotype in tuberculosis patients. Immunology. 2004;111:100–6.CrossRefGoogle Scholar
  28. 28.
    Maeda T, Yamada H, Nagamine R, Shuto T, Nakashima Y, Hirata G, et al. Involvement of CD4+,CD57+ T cells in the disease activity of rheumatoid arthritis. Arthritis Rheum. 2002;46:379–84.CrossRefGoogle Scholar
  29. 29.
    Imberti L, Sottini A, Signorini S, Gorla R, Primi D. Oligoclonal CD4+ CD57+ T-cell expansions contribute to the imbalanced T-cell receptor repertoire of rheumatoid arthritis patients. Blood. 1997;89:2822–32.PubMedGoogle Scholar
  30. 30.
    Zhang Z, Wu N, Lu Y, Davidson D, Colonna M, Veillette A. DNAM-1 controls NK cell activation via an ITT-like motif. J Exp Med. 2015;212:2165–82.CrossRefGoogle Scholar
  31. 31.
    Oosterhoff D, van de Weerd G, van Eikenhorst G, de Gruijl TD, van der Pol LA, Bakker WA. Hematopoietic cancer cell lines can support replication of Sabin poliovirus type 1. Biomed Res Int. 2015;2015:358462.CrossRefGoogle Scholar
  32. 32.
    Sunami Y, Sato E, Ichikawa K, Yasuda H, Komatsu N. Hemorrhagic colitis caused by dasatinib following cytomegalovirus enterocolitis in a patient with chronic myelogenous leukemia in the second chronic phase. Rinsho Ketsueki. 2011;52:282–6.PubMedGoogle Scholar
  33. 33.
    Kreutzman A, Ladell K, Koechel C, Gostick E, Ekblom M, Stenke L, et al. Expansion of highly differentiated CD8+ T-cells or NK-cells in patients treated with dasatinib is associated with cytomegalovirus reactivation. Leukemia. 2011;25:1587–97.CrossRefGoogle Scholar
  34. 34.
    Tanaka H, Nakashima S, Usuda M. Rapid and sustained increase of large granular lymphocytes and rare cytomegalovirus reactivation during dasatinib treatment in chronic myelogenous leukemia patients. Int J Hematol. 2012;96:308–19.CrossRefGoogle Scholar
  35. 35.
    Qiu ZY, Xu W, Li JY. Large granular lymphocytosis during dasatinib therapy. Cancer Biol Ther. 2014;15:247–55.CrossRefGoogle Scholar
  36. 36.
    van Bergen J, Kooy-Winkelaar EM, van Dongen H, van Gaalen FA, Thompson A, Huizinga TW, et al. Functional killer Ig-like receptors on human memory CD4+ T cells specific for cytomegalovirus. J Immunol. 2009;182:4175–82.CrossRefGoogle Scholar
  37. 37.
    Fei F, Yu Y, Schmitt A, Rojewski MT, Chen B, Gotz M, et al. Dasatinib inhibits the proliferation and function of CD4+ CD25+ regulatory T cells. Br J Haematol. 2009;144:195–205.CrossRefGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2018

Authors and Affiliations

  • Naoki Watanabe
    • 1
  • Tomoiku Takaku
    • 1
    Email author
  • Kazuyoshi Takeda
    • 2
    • 3
  • Shuichi Shirane
    • 1
  • Tokuko Toyota
    • 1
  • Michiaki Koike
    • 4
  • Masaaki Noguchi
    • 5
  • Takao Hirano
    • 6
  • Hiroshi Fujiwara
    • 7
  • Norio Komatsu
    • 1
  1. 1.Department of HematologyJuntendo University School of MedicineTokyoJapan
  2. 2.Division of Cell Biology, Biomedical Research Center, Graduate School of MedicineJuntendo UniversityTokyoJapan
  3. 3.Department of Biofunctional Microbiota, Graduate School of MedicineJuntendo UniversityTokyoJapan
  4. 4.Department of HematologyJuntendo Shizuoka HospitalShizuokaJapan
  5. 5.Department of HematologyJuntendo Urayasu HospitalChibaJapan
  6. 6.Department of HematologyJuntendo Nerima HospitalTokyoJapan
  7. 7.Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime University Graduate School of MedicineEhime UniversityToonJapan

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