Mutated Regions Of Nucleophosmin1Elicit CD8~+T-cell Responses In Patients With Acute Myeloid Leukemia

This thesis will include two parts: Part I: To induce specific T lymphocytes responsesfor NPM1mutderived peptides by dendritic cells loaded with these peptides in vitro; Part II:To detect the peptide specific T cells in peripheral blood of NPM1mutpositive acutemyeloid leukemia patients.【Objective】Stimulating lymphocytes from healthy donors by DCs loaded with NPM1mutpeptidesin vitro in order to induce specific cellular immune responses against the peptides. Anddetecting the NPM1mutpeptide specific T-cell in peripheral blood of patients with NPMmutpositive acute myeloid leukemia patients (AML). Therefore, to provide a theoretical basisfor immune therapy of AML patients.【Methods】1. NPM1mutation detection: our hospital newly diagnosed AML patients underwentgene mutations detection routinely. The process basically includes several steps as follows:extracting genomic DNA from patient’s bone marrow or peripheral blood mononuclearcells, amplification of NPM1gene in exon12by general polymerase chain reaction (PCR)and direct sequencing of PCR products.2. Human leukocyte antigen (HLA) genotyping: detecting HLA-A, B, C, DRB1,DQB1five loci by polymerase chain reaction-sequencing based typing (PCR-SBT).3. Samples’ collection and preservation: acquiring peripheral blood from HLA-A*0201or A*1101positive healthy donors and NPM1mut+AML patients in completeremission. Ficoll-Paque gradient density centrifuged PBMCs were cryopreserved in liquidnitrogen.4. CD8+T cell epitopes prediction and peptides synthesis: the entire amino acidsequences of the NPM1wild-type protein as well as of the mutated NPM1types A and D were screened for HLA-A*0201, A*1101or A*2402-binding T-cell epitopes using theNet-CTL1.2, Rankpep, and SYFPEITHI software programs. The nonamer peptides withthe highest scores were produced by a solid-phase synthesizer and purified (>95%).5. DCs incubation:3ml PBMCs suspention (3~5×106/ml) in RPMI1640mediumwere seeded in six-well plates to enrich the monocyte fraction. After incubating2hours at37℃and5%CO2, nonadherent cells were removed, and adherent cells were subsequentlycultured for5days with granulocyte macrophage colony-stimulating factor (GM-CSF) andIL-4in serum-free medium to induce monocytes differentiate to immature dendritic cells..Then cells were cultured in medium containing GM-CSF, IL-4, IL-6, IL-1β, TNF-α, PGE2and cultured for2days to induce DCs maturation. The DCs were harvested after7dayswhich would be used as antigen-presenting cells (APC) and tested their surface antigens byflow cytometric analysis.6. Generation of NPM1-specific cytotoxic T cells: DCs were pulsed with differentNPM1peptides while set a nonpeptide pulsed group. Then DCs were irradiated with40Gy.Peptide-pulsed DCs were then coincubated with the thawed itself PBMCs (1:10) inserum-free medium at37°C and5%CO2, maintained by IL-2and IL-7. Lymphocytesrestimulated by peptide-pulsed DCs on days7.7. ELISPOT analysis and intracellular staining: peptide-stimulated T cells wereweekly basis on days7and14tested by ELISPOT and intracellular staining method. Thesetwo methods both detected the secretion of IFN-γ of cytotoxic T cells. T2cells were usedas target cells, and terms on HLA-A*1101positive donors would use themselves DCs astargets. Then we divided into three groups according to target cells weather to be pulsedwith peptide and the kind of peptide. Respectively, negative control group (nonpeptidepulsed), NPM1wildpeptide pulsed cells group, NPM1mutpeptides pulsed cells group.Peripheral blood samples of NPM1mut+AML patients were thawed and tested by ELISPOTanalysis after rest overnight in serum free medium at37°C and5%CO2.【Results】1. In the Han Chinese population, the three most common alleles of HLA-A loci wereA*1101, A*2402, A*0201allele.2. Sreened for HLA-A*0201, A*1101or A*2402-binding T-cell epitopes of wild andmutated NPM1proteins by aforementioned three different algorithms. Eventually,HLA-A*0201restricted wild NPM1amino acid sequences (DLWQWRKSL), mutated NPM1-A/D amino acid sequences (AIQDLCLAV), and HLA-A*1101restricted mutatedNPM1-A/D amino acid sequences (AVEEVSLRK) were synthesized. There were noproper epitopes for HLA-A*1101restricted wild NPM1protein and HLA-A*2402restricted neither the wild nor mutated.3. Expression of surface antigens of DCs (CD14, CD80, CD83, CD86, HLA-DR) ondays7were2.6%,43.4%,7.8%,99.9%,67.1%, respectively, which was consistent withDCs phenotype.4. On days10of DCs incubated with themselves PBMCs, we saw a large number ofcells proliferation, especially in the NPM1mutpeptide pulsed group. On days7, ELISPOTanalysis results of all samples were negative; On days14,3cases’ ELISPOT results werepositive for the mutated peptide holes，and the wild peptide holes were negative; theNPM1mutpeptide-specific T lymphocytes’ positive rate [=(average number of spotsmutated peptide holes-the average number of spots negative control group)/totallymphocytes per hole] was about1/2500. Intracellular staining showed that (Gating toCD3+CD8+cells): in the aforementioned3cases, the positive rate of CD8+IFN-γ+cells ofthe mutant peptide group was higher than either the wild peptide group or negative controlgroup, but there was no difference between the wild and the negative.5. The6peripheral blood of patients with NPM1mut+AML were performed ELISPOTanalysis, and results showed only1case (16.7%) with a approaching positive result.【Conclusions】NPM1mutpeptide pulsed DCs can stimulate itself PBMCs from healthy donors in vitroto produce mutated NPM1specific T lymphocytes, and it is expected to be used as immunetherapy of AML. The mutated NPM1specific CTL in NPM1mut+AML patients are almostundetectable that indicates immune system have been comprised because of chemotherapyand disease, and can not produce sufficient responses to antigens. Perhaps AML patientscan accept CTL transfusion from their relatives. Our study results supply an experimentalbasis for cellular immunotherapy of AML.