Contributions
Type: Publication Only
Background
Stimulating lymphocytes from healthy donors by DCs loaded with NPM1mutpeptides in vitro in order to induce specific cellular immune responses against the peptides.
Aims
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. newly diagnosed patients of AML underwent gene mutations screening routinely.
2. peripheral blood mononuclear cells (PBMCs) were isolatedfrom healthy donors with HLA-A* 0201 or A*1101 and NPM1mut positive AML patients in complete remission.
3. Inducing differentiation of PBMCs into DCs
4. Generation of NPM1-specific cytotoxic T cells
5. ELISPOT analysis and intracellular staining
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.
Summary
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.
Keyword(s): Acute myeloid leukemia, CD8 T cells, Mutation
Type: Publication Only
Background
Stimulating lymphocytes from healthy donors by DCs loaded with NPM1mutpeptides in vitro in order to induce specific cellular immune responses against the peptides.
Aims
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. newly diagnosed patients of AML underwent gene mutations screening routinely.
2. peripheral blood mononuclear cells (PBMCs) were isolatedfrom healthy donors with HLA-A* 0201 or A*1101 and NPM1mut positive AML patients in complete remission.
3. Inducing differentiation of PBMCs into DCs
4. Generation of NPM1-specific cytotoxic T cells
5. ELISPOT analysis and intracellular staining
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.
Summary
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.
Keyword(s): Acute myeloid leukemia, CD8 T cells, Mutation