Characterization and dynamics of specific T cells against nucleophosmin-1 (NPM1)-mutated peptides in patients with NPM1-mutated acute myeloid leukemia

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.


Patients and samples
We have enrolled a cohort of 31 adult patients (median age 56 years, range 19-75), affected with NPM1mutated AML, and 11 healthy volunteers. All the enrolled patients, except for one elderly subject (Pt 29) who underwent hypomethylating treatment with 5-azacitidine, received intensive remission induction chemotherapy.
Either bone marrow mononuclear cells (BMMCs) or peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (Axis-Shield PoC AS, Oslo, Norway), washed twice in RPMI1640 culture medium, and used either fresh or cryopreserved for immunological analyses.

NPM1-mutated-derived peptides
NPM1-mutated peptides have been synthesized by Biosense to a minimum purity of 70% (immunograde purity) and confirmed by mass spectrometry. All the 18 peptides tested were utilized as leukemia-specific antigen stimulation for all the immunological assays performed in this study, either as mixtures or as individual peptides.

Enzyme-linked immunospot (ELISPOT) assay
Cells were counted by the use of automated cell counters (AcT8, Beckman Coulter), resuspended at the appropriate cell density in RPMI1640/fetal calf serum 10% (Invitrogen) and cultured at 37° C in a humidified 5% CO2 atmosphere in 96-well bottom plates coated with anti-IFNγ monoclonal antibodies (Mabtech, Nacka Strand, Sweden). 10 A total of 2.5 × 10 5 cells/well were stimulated for 20 hours either with different mixed pools of NPM1-mutated peptides (each peptide used at a final concentration of 50 μg/ml) or with each peptide individually. Unstimulated BMMCs or PBMCs were used as negative controls, whereas anti-CD3 antibody (Mabtech) was added to positive control wells at a final concentration of 5 μg/ ml. After incubation, biotinylated secondary antibody (Mabtech, 0.5 µg/ml) was added, and plates were then processed according to standard procedures [10].
The number of spot forming cells (SFCs) per well was quantified using an automated ELISPOT counter (AID-GmbH, Strassberg, Germany). All test conditions were carried out in triplicate and results for individual time-points were calculated as a median value of SFCs obtained after antigenic stimulations compared to control wells and expressed as number of SFCs on 10 6 PBMCs or BMMCs. Results were considered positive if the number of SFCs/10 6 cells in NPM1-mutated antigen-stimulated wells was 2-fold higher than that in negative control wells, and there were at least 60 SFCs/10 6 cells, according to Cancer Immunoguiding Program guidelines (www.cimt. eu/cms/diskfiles/download/7/418244ec26c905deaf7b1e9 cc38d004f/cip_guidelines.pdf) Mann-Whitney U test or Wilcoxon signed rank test were performed to compare differences between continuous variables, whereas Chisquare analysis and Fisher's exact test were performed for categorical variables. A value of p <0.05 was considered statistically significant.

Cytokine secretion assay (CSA)
In summary, 1 × 10 6 PBMCs or BMMCs were cultured at 37° C and stimulated for 3 hours with the 18 NPM1mutated peptides comprehensive mixture, as used for Elispot assay. In parallel, for each analysis, unstimulated and PHAstimulated PBMCs or BMMCs were used as negative and positive controls, respectively. Subsequently, samples were first labelled with the Catch Reagent, and thereafter with the Detection Reagent, according to the manufacturer's instructions, in order to detect cytokine-secreting cells.
Cells were acquired on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA), and analyzed by the use of CellQuest (BD Biosciences) and Summit software (Dako Colorado, Fort Collins, CO). Either CD8+ or CD4+ T cells were gated on CD3+ events after passing through a small lymphocyte gate and frequencies of antigen-reactive EM or CM T cells were calculated as median differences compared to unstimulated controls.

Minimal residual disease (MRD) monitoring for NPM1-mutated transcripts by reversetranscriptase quantitative polymerase chain reaction (RQ-PCR)
One microgram of total RNA, extracted from BMMCs, was reverse transcribed in 20 μl reverse-transcriptase reaction. Real-time quantitative PCR was carried out on cDNA on a LightCycler 480 (ROCHE) using the TaqMan method.
NPM1-mutated transcripts levels were compared to expression of ABL1 (pre-developed assay reagents), used as housekeeping gene. The absolute quantification of NPM1mutated was obtained using a standard curve designed with the fit point method.