Abstract
Nucleophosmin(NPM1)-mutated protein, a leukemia-specific antigen, represents an ideal target for AML immunotherapy. We investigated the dynamics of NPM1-mutated-specific T cells on PB and BM samples, collected from 31 adult NPM1-mutated AML patients throughout the disease course, and stimulated with mixtures of 18 short and long peptides (9-18mers), deriving from the complete C-terminal of the NPM1-mutated protein. Two 9-mer peptides, namely LAVEEVSLR and AVEEVSLRK (13.9–14.9), were identified as the most immunogenic epitopes. IFNγ-producing NPM1-mutated-specific T cells were observed by ELISPOT assay after stimulation with peptides 13.9–14.9 in 43/85 (50.6%) PB and 34/80 (42.5%) BM samples. An inverse correlation between MRD kinetics and anti-leukemic specific T cells was observed. Cytokine Secretion Assays allowed to predominantly and respectively identify Effector Memory and Central Memory T cells among IFNγ–producing and IL2–producing T cells. Moreover, NPM1-mutated-specific CTLs against primary leukemic blasts or PHA-blasts pulsed with different peptide pools could be expanded ex vivo from NPM1-mutated AML patients or primed in healthy donors. We describe the spontaneous appearance and persistence of NPM1-mutated-specific T cells, which may contribute to the maintenance of long-lasting remissions. Future studies are warranted to investigate the potential role of both autologous and allogeneic adoptive immunotherapy in NPM1-mutated AML patients.
Highlights
Nucleophosmin (NPM1) gene mutations, occurring in approximately 30% of adult acute myeloid leukemia (AML) cases, and in 50–60% of AML cases with a normal karyotype, represent one of the most frequent molecular lesions observed in AML [1,2,3]
We performed IFNγ-Enzyme-linked immunospot (ELISPOT) assays to investigate the occurrence of NPM1-mutated-specific T cells in a total of 137 peripheral blood (PB) and 80 bone marrow (BM) samples (Table 1), collected at different time points, from 31 adults, affected with NPM1mutated AML (Figure 1)
In the initial phase of the study, ELISPOT assays, carried out after antigenic stimulation with a comprehensive mixture containing all of the 18 NPM1mutated peptides, documented NPM1-mutated-specific T cells producing IFNγ in 34/52 (65.4%) PB samples, obtained from the first 17 patients enrolled in the study (Figure 1A)
Summary
Nucleophosmin (NPM1) gene mutations, occurring in approximately 30% of adult acute myeloid leukemia (AML) cases, and in 50–60% of AML cases with a normal karyotype, represent one of the most frequent molecular lesions observed in AML [1,2,3]. NPM1 mutations are specific, being almost exclusively restricted to AML, and usually expressed in the entire leukemic population [1, 4, 5]. NPM1 cytoplasmic dislocation may favor protein processing and degradation pathways, presumably leading to more efficient human leukocyte antigen (HLA) presentation [6]. None of the normal human sequences present in databanks match those of the 11 C-terminal residues of the NPM1 mutants, suggesting that this aminoacidic sequence may serve as a leukemia-specific antigen [6]. Based upon the above mentioned biological characteristics, NPM1mutated protein may be considered an ideal target antigen for AML immunotherapy [7]
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