Mutated Regions of Nucleophosmin 1 Elicit CD8+ T-Cell Responses in Patients with Acute Myeloid Leukemia
Authors: Sun Aining , Cheng Zhen , Chen Guanghua , Yang Xiaofei , Depei Wu 
Published: October 2019

Abstract
Objective
Stimulating lymphocytes from healthy donors by DCs loaded with NPM1mut peptides in vitro in order to induce specific cellular immune responses against the peptides. And detecting the NPM1mut peptide specific T-cell in peripheral blood of patients with NPM1mut positive acute myeloid leukemia (AML), to provide a theoretical basis for immune therapy of AML.
Methods
1. NPM1 mutation detection: newly diagnosed patients of AML underwent gene mutations screening routinely.
2. Samples collection: peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood collected from healthy donors with HLA-A* 0201 or A*1101 and NPM1mut positive AML patients in complete remission.
3. Inducing differentiation of PBMCs into DCs: non-adherent cells were removed from PBMCs suspension, and then adherent cells were subsequently cultured for 5 days with GM-CSF and IL-4 to induce differentiation. Then cells were cultured in medium containing GM-CSF, IL-4, IL-6, IL-1β, TNF-α, and PGE2 for another 2 days to induce maturation.
4. Generation of NPM1-specific cytotoxic T cells: DCs were pulsed with different NPM1 peptides followed by irradiation with 40 GY. The non-peptide pulsed DCs group was set as negative control. The above mentioned DCs were then co-incubated with their own PBMCs, maintained by IL-2 and IL-7. On day 7 of co-incubation, the same treated DCs as mentioned above were added again to restimulate lymphocytes.
5. ELISPOT analysis and intracellular staining: peptide-stimulated T cells were tested by ELISPOT and intracellular staining method on day 7 and 14 . These two methods both detected the secretion of IFN-γ of cytotoxic T cells. T2 cells were used as target cells, and in terms of HLA-A*1101 positive donors, their own DCs were used as targets.
Results
1. In the Han Chinese population, the most common alleles of HLA-A loci were A* 1101, A*2402, and A*0201 allele.
2. Through prediction of the aforementioned software, HLA-A*0201 restricted wild type NPM1 amino acid sequences (DLWQWRKSL), mutated NPM1-A/D amino acid sequences (AIQDLCLAV), and HLA-A*1101 restricted mutated NPM1-A/D amino acid sequences (AVEEVSLRK) were synthesized. There were no proper epitopes for either HLA-A*1101 restricted wild type NPM1 protein or HLA-A*2402 restricted wild type and mutated NPM1 protein.
3. Expression of surface antigens of DCs on day 7 were as follows: CD14(2.6%), CD80(43.4%), CD83(7.8%), CD86(99.9%), HLA-DR(67.1%), which was consistent with DCs phenotype.
4. About 10 days after DCs were co-incubated with their own PBMCs, the number of lymphocytes increased significantly, especially in the NPM1mut peptide pulsed group. On day 7 of co-incubation, ELISPOT analysis results of all samples were negative. However, 3 cases’ ELISPOT results were positive for the mutated peptide holes on day 14, in contrast, the results of the wild type peptide holes were negative. The NPM1mut peptide-specific T lymphocytes positive rate [=(average number of spots in mutated peptide holes-the average number of spots in negative control holes)/total lymphocytes per hole] was about 1/2500. Intracellular staining showed that in the aforementioned 3 cases, the proportion of CD8+IFN-γ +cells of the mutant peptide group was higher than either the wild type peptide group or negative control group, but there was no difference between the later two groups.
5. 6 peripheral blood samples of patients with NPM1mut +AML were performed ELISPOT analysis, with only 1 case (16.7%) showing a approaching positive result.
Conclusions
NPM1mut peptide pulsed DCs can stimulate their own PBMCs from healthy donors in vitro to produce mutated NPM1 specific T lymphocytes, and it is expected to be used as immune therapy of AML. The mutated NPM1 specific CTL in NPM1mut+AML patients are almost undetectable that indicates immune system have been comprised because of chemotherapy and disease, and can not responses to antigens efficiently. Perhaps AML patients can accept CTL transfusion from their relatives. Our study provide an experimental basis for cellular immunotherapy of AML.

Disclosures
No relevant conflicts of interest to declare.