Effect of epitopes derived from the mutated region of cytoplasmatic nucleophosmine 1 (NPM1) on CD4+ and CD8+ T-cell responses in patients with acute myeloid leukemia.
6567 Background: Mutations of the nucleophosmin gene (NPM1mut) are one of the most frequent molecular alterations in AML and constitute an important prognostic marker. The impact of NPM1mut on leukemogenesis and progression remains to be elucidated. Immune responses against NPM1mut might contribute to the favourable prognosis of AML patients with NPM1mut. Therefore, we examined T cell responses against NPM1mut. Methods: NPM1 wildtype as well as NPM1mut were screened for HLA-A*0201 binding T cell epitopes with the help of different algorithm programs. Ten peptides with most favourable characteristics were tested with ELISpot analysis for interferon-γ and granzyme B in 33 healthy volunteers and 30 AML patients. Tetramer assays against most interesting epitopes were performed and chromium release assays were used to show the cytotoxicity of peptide-specific CD8+ T cells. Moreover, HLA-DR-binding epitopes were used to test the role of CD4+ T cells in NPM1 immunogenicity. Results: Two epitopes (#1 and #3) derived from NPM1mut induced CD8+ T cell responses in a high frequency. In healthy volunteers, immune responses were detected in 39%/18% against #1 and #3, and in 33%/44% of NPM1mut AML patients against #1 and #3. NPM1-peptide primed effector T cells showed specific lysis of pulsed T2 cells as well as leukemic blasts in chromium release assays. In tetramer assays a significant CD8+ T cell population could be detected. To obtain a robust and continuous T cell reaction, the help of CD4+ T cells is indispensable. Therefore, we investigated the increase of CD8+ T cell responses by the activation of CD4+ T cells stimulated with longer peptides called overlapping peptides (OL). Potent HLA-DR epitopes were predicted and several favourable peptides (OL 1 to 8) were synthesized. OL8 showed favourable results to activate both CD8+ and CD4+ T cells. Conclusions: Taken together, NPM1mut represents a candidate for immunotherapeutic approaches and we hypothesize that it is also potentially involved in immunogenic rejection of NPM1mut leukemic blasts. Therefore, NPM1mut is a promising target structure for specific immunotherapies in AML patients.