INTRODUCTION
Acute myeloid leukemia (AML) is a heterogeneous group of disorders characterized by malignant clonal proliferation of myeloid blast cells in the bone marrow (BM) and peripheral blood, leading to cytopenia, infections, and bleeding 1,2. Although many therapeutic interventions have been explored to treat patients with AML, chemotherapeutic regimens remain a crucial therapy component for these patients 3. In the AML treatment chemotherapy guideline, the two antineoplastic agents, fludarabine and busulfan, are well-known. Fludarabine shows promising results in treating relapsed/refractory patients with AML; however, the neurotoxicity effect of this agent limits the use of high-dose fludarabine4. Despite its lower costs and acceptable efficiency over the decades, the significant side effects of busulfan, including mucositis of grade 2 or higher, led to the displacement of this agent with more expensive but secure monoclonal antibody imatinib5. To date, NK cell-based immunotherapy is one of the most current innovative immunotherapeutic techniques, unleashing the immunological suppression of NK cells to attack a variety of malignancies 6. In AML, dysfunctional NK cells or immunosuppressive features of AML cells and their prognostic relevance justify using NK cell-based immunotherapy to restore impaired NK cell cytotoxicity against AML 7. Meanwhile, treatment with busulfan and fludarabine inactive NK cells (PMID: 2933271) with unknown mechanism. Another defined mechanism that makes weak NK cells, as well as T cells in patients with AML, is overexpression of inhibitory immune checkpoint molecules such as programmed cell death ligand-1 (PD-L1) and PD-L2 up-regulated in blasts 8. Furthermore, Heat shock protein 70 KDa (Hsp70) is an essential component of the protein folding system called chaperones and protects the cells from stress-induced damage 5. Hsp70 as an antigenic peptide9 can be used as a tumor-specific vaccine10. Furthermore, Hsp70 induces the release of pro-inflammatory cytokines from innate immune cells, increasing the expression of costimulatory molecules 11. In addition, Hsp70 activates the NK cell cytotoxic effects against the Hsp70 representing tumor cells 12. Despite promising outcomes resulting from investigations performed on PD-1 blocker base immunotherapy on solid tumors, the efficacy of this approach is not studied on hematologic malignancies. Therefore, in the present study, we assessed the combined effect of PD-1 blocker and Hsp70 on the activation of NK cells derived from patients with relapsed AML under treatment of Busulfan and fludarabine.