Hsp70, in Combination with IL-15 and PD-1 Blocker, Interferes with The Induction of Cytotoxic NK Cells in Relapsed Acute Myeloid Leukemia Patients

Document Type : Original Article


1 Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran

2 Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

3 Cellular and Molecular Research Centre, Iran University of Medical Sciences, Tehran, Iran

4 Taleghani Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Kurdistan, Iran

6 Department of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran

7 Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran


Objective: Natural killer (NK) cells are critical immune cells for acute myeloid leukemia (AML) targeting. However,
little is known about the relationship between using checkpoint inhibitors and heat shock protein 70 (Hsp70) as NK cell
activators to control AML. Therefore, the study aims to find the best formulation of Hsp70, human PD-1 (Programmed
cell death protein 1) blocker, and interleukin 15 (IL-15) to activate NK cells against AML.
Materials and Methods: In this experimental study, the NK cells were isolated from mononuclear cells (MNCs) by
using magnetic activation cell sorting (MACS) and were activated using the different combinations of Hsp70, PD-1
blocker, and IL-15 and then followed by immunophenotyping, functional assays to estimate their killing potential, and
evaluation of expression pattern of PRF1, PIK3CB, PD-1, AKT-1, FAS-L, TRAIL, and GER A and B.
Results: The expression of PD-1 was significantly (P<0.05) reduced after NK cell activation by the different formulas of
IL-15, Hsp70, and PD-1 blocker. The expression of NKG2A in the treated NK cells was reduced particularly in the IL-15
(P<0.01) and IL-15+PD-1 blocker (P<0.05) groups. The addition of Hsp70 increased its expression. The cytotoxic effect
of NK cells increased in all groups, especially in IL-15+PD-1 blocker besides increasing interferon-gamma (IFN-γ),
Granzymes, and perforin expression (P<0.05). All IL-15+PD-1 blocker group changes were associated with the upregulation
of PIK3CB and AKT-1 as key factors of NK cell activation. The presence of Hsp70 reduced IFN-γ releasing,
and down-regulation of PIK3CB, AKT-1, Granzymes, and Perforin (P<0.05).
Conclusion: We suggested the combination of IL-15 and PD-1 blocker could enhance the killing potential of AMLNK
cells. Moreover, Hsp70 in combination with IL-15 and PD-1 blocker interferes activation of AML-NK cells through
unknown mechanisms.


Main Subjects

  1. Ghazawi FM, Le M, Cyr J, Netchiporouk E, Rahme E, Alakel A, et al. Analysis of acute myeloid leukemia incidence and geographic distribution in Canada from 1992 to 2010 reveals disease clusters in Sarnia and other industrial US border cities in Ontario. Cancer. 2019; 125(11): 1886-1897.
  2. Zhang C, Lam SSY, Leung GMK, Tsui SP, Yang N, Ng NKL, et al. Sorafenib and omacetaxine mepesuccinate as a safe and effective treatment for acute myeloid leukemia carrying internal tandem duplication of Fms-like tyrosine kinase 3. Cancer. 2020; 126(2): 344-353.
  3. Kantarjian HM, Jabbour EJ, Garcia-Manero G, Kadia TM, Di- Nardo CD, Daver NG, et al. Phase 1/2 study of DFP-10917 administered by continuous intravenous infusion in patients with recurrent or refractory acute myeloid leukemia. Cancer. 2019; 125(10): 1665-1673.
  4. Abdolkarimi B, Zareifar S, Karimi M, Salajegheh P. Management of refractory/relapsed acute leukemia with heart limitation by anthracycline-free chemotherapy regimens in pediatric patients: New hypothesis and new approach. MEJC. 2018; 9(2): 77-84.
  5. Rasor B, Dickerson T, Zhao Q, Elder P, Brammer JE, Larkin K, et al. Comparison of fixed dose reduced-intensity conditioning with fludarabine and busulfan to PK-guided busulfan AUC (FluBu4K) in hematopoietic stem cell transplant for AML/MDS. Leuk Lymphoma. 2021; 62(4): 944-951.
  6. Ciurea SO, Kongtim P, Soebbing D, Trikha P, Behbehani G, Rondon G, et al. Decrease post-transplant relapse using donorderived expanded NK-cells. Leukemia. 2022; 36(1): 155-164.
  7. Martín-Antonio B, Suñe G, Perez-Amill L, Castella M, Urbano- Ispizua A. Natural killer cells: angels and devils for immunotherapy. Int J Mol Sci. 2017; 18(9): 1868.
  8. Xu J, Niu T. Natural killer cell-based immunotherapy for acute myeloid leukemia. J Hematol Oncol. 2020; 13(1): 167.
  9. Shevtsov M, Multhoff G. Heat shock protein–peptide and HSPbased immunotherapies for the treatment of cancer. Front Immunol. 2016; 7: 171.
  10. Gong J, Zhang Y, Durfee J, Weng D, Liu C, Koido S, et al. A heat shock protein 70-based vaccine with enhanced immunogenicity for clinical use. J Immunol. 2010; 184(1): 488-496.
  11. Ferat-Osorio E, Sánchez-Anaya A, Gutiérrez-Mendoza M, Boscó-Gárate I, Wong-Baeza I, Pastelin-Palacios R, et al. Heat shock protein 70 down-regulates the production of toll-like receptor- induced pro-inflammatory cytokines by a heat shock factor- 1/constitutive heat shock element-binding factor-dependent mechanism. J Inflamm (Lond). 2014; 11: 19.
  12. Specht HM, Ahrens N, Blankenstein C, Duell T, Fietkau R, Gaipl US, et al. Heat shock protein 70 (Hsp70) peptide activated natural killer (NK) cells for the treatment of patients with non-small cell lung cancer (NSCLC) after radiochemotherapy (RCTx)–from preclinical studies to a clinical phase II trial. Front Immunol. 2015; 6: 162.
  13. Yang L, Shen M, Xu LJ, Yang X, Tsai Y, Keng PC, et al. Enhancing NK cell-mediated cytotoxicity to cisplatin-resistant lung cancer cells via MEK/Erk signaling inhibition. Sci Rep. 2017; 7(1): 7958.
  14. Niu C, Li M, Zhu S, Chen Y, Zhou L, Xu D, et al. PD-1-positive natural killer cells have a weaker antitumor function than that of PD-1-negative natural killer cells in lung cancer. Int J Med Sci. 2020; 17(13): 1964-1973.
  15. Ewen CL, Kane KP, Bleackley RC. A quarter century of granzymes. Cell Death Differ. 2012; 19(1): 28-35.
  16. Chung YM, Khan PP, Wang H, Tsai W-B, Qiao Y, Yu B, et al. Sensitizing tumors to anti-PD-1 therapy by promoting NK and CD8+ T cells via pharmacological activation of FOXO3. J Immunother Cancer. 2021; 9(12): e002772.
  17. Ambrose AR, Hazime KS, Worboys JD, Niembro-Vivanco O, Davis DM. Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles. Proc Natl Acad Sci USA. 2020; 117(38): 23717-23720.
  18. Zhu Y, Huang B, Shi J. Fas ligand and lytic granule differentially control cytotoxic dynamics of natural killer cell against cancer target. Oncotarget. 2016; 7(30): 47163-47172.
  19. Ali AK, Nandagopal N, Lee SH. IL-15–PI3K–AKT–mTOR: a critical pathway in the life journey of natural killer cells. Front Immunol. 2015; 6: 355.
  20. Ruiz-García R, Vargas-Hernández A, Chinn IK, Angelo LS, Cao TN, Coban-Akdemir Z, et al. Mutations in PI3K110δ cause impaired natural killer cell function partially rescued by rapamycin treatment. J Allergy Clin Immunol. 2018; 142(2): 605-617. e7.
  21. McDermott DF, Atkins MB. PD-1 as a potential target in cancer therapy. Cancer Med. 2013; 2(5): 662-673.
  22. Wojtukiewicz MZ, Rek MM, Karpowicz K, Górska M, Polityńska B, Wojtukiewicz AM, et al. Inhibitors of immune checkpoints— PD-1, PD-L1, CTLA-4—new opportunities for cancer patients and a new challenge for internists and general practitioners. Cancer Metastasis Rev. 2021; 40(3): 949-982.
  23. Clara JA, Childs RW. Harnessing natural killer cells for the treatment of multiple myeloma. Semin Oncol. 2022; 49(1): 69- 85.
  24. Costa F, Marchica V, Storti P, Malavasi F, Giuliani N. PD-L1/ PD-1 axis in multiple myeloma microenvironment and a possible link with CD38-mediated immune-suppression. Cancers (Basel). 2021; 13(2): 164.
  25. Albakova Z, Armeev GA, Kanevskiy LM, Kovalenko EI, Sapozhnikov AM. HSP70 multi-functionality in cancer. Cells. 2020; 9(3): 587.
  26. Murphy ME. The HSP70 family and cancer. Carcinogenesis. 2013; 34(6): 1181-1188.
  27. Reikvam H, Hatfield KJ, Ersvær E, Hovland R, Skavland J, Gjertsen BT, et al. Expression profile of heat shock proteins in acute myeloid leukaemia patients reveals a distinct signature strongly associated with FLT3 mutation status–consequences and potentials for pharmacological intervention. Br J Haematol. 2012; 156(4): 468-480.
  28. Ryningen A, Ersvær E, Øyan AM, Kalland KH, Vintermyr OK, Gjertsen BT, et al. Stress-induced in vitro apoptosis of native human acute myelogenous leukemia (AML) cells shows a wide variation between patients and is associated with low BCL-2: Bax ratio and low levels of heat shock protein 70 and 90. Leuk Res. 2006; 30(12): 1531-1540.
  29. Lobinger D, Gempt J, Sievert W, Barz M, Schmitt S, Nguyen HT, et al. Potential role of Hsp70 and activated NK cells for prediction of prognosis in glioblastoma patients. Front Mol Biosci. 2021; 8: 669366.
  30. Elsner L, Flügge PF, Lozano J, Muppala V, Eiz-Vesper B, Demiroglu SY, et al. The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells. J Cell Mol Med. 2010; 14(4): 992-1002.
  31. Rad HA, Basirat Z, Mostafazadeh A, Faramarzi M, Bijani A, Nouri HR, et al. Evaluation of peripheral blood NK cell subsets and cytokines in unexplained recurrent miscarriage. J Chin Med Assoc. 2018; 81(12): 1065-1070.
  32. Backström E, Kristensson K, Ljunggren HG. Activation of natural killer cells: underlying molecular mechanisms revealed. Scand J Immunol. 2004; 60(1-2): 14-22.
  33. Moynagh PN. IL-15 in autoimmunity and cancer: O-tu-b or not O-tu-b? Nat Immunol. 2019; 20(7): 780-782.
  34. Nandagopal N, Ali AK, Komal AK, Lee SH. The critical role of IL-15–PI3K–mTOR pathway in natural killer cell effector functions. Front Immunol. 2014; 5: 187.
  35. Fehniger TA, Caligiuri MA. Interleukin 15: biology and relevance to human disease. Blood. 2001; 97(1): 14-32.
  36. Calvo T, Reina-Ortiz C, Giraldos D, Gascón M, Woods D, Asenjo J, et al. Expanded and activated allogeneic NK cells are cytotoxic against B-chronic lymphocytic leukemia (B-CLL) cells with sporadic cases of resistance. Sci Rep. 2020; 10(1): 19398.
  37. Shevtsov M, Pitkin E, Ischenko A, Stangl S, Khachatryan W, Galibin O, et al. Ex vivo Hsp70-activated NK cells in combination with PD-1 inhibition significantly increase overall survival in preclinical models of glioblastoma and lung cancer. Front Immunol. 2019; 10: 454.
  38. Zeng Y, Lv X, Du J. Natural killer cell‑based immunotherapy for lung cancer: Challenges and perspectives (review). Oncol Rep. 2021; 46(5): 232.
  39. Thomas X, Campos L, Le QH, Guyotat D. Heat shock proteins and acute leukemias. Hematology. 2005; 10(3): 225-235.
  40. Reikvam H, Nepstad I, Sulen A, Gjertsen BT, Hatfield KJ, Bruserud Ø. Increased antileukemic effects in human acute myeloid leukemia by combining HSP70 and HSP90 inhibitors. Expert Opin Investig Drugs. 2013; 22(5): 551-563.