Neutrophil extracellular traps arm DC vaccination against NPM-mutant myeloproliferation

  1. Claudio Tripodo
  2. Barbara Bassani
  3. Elena Jachetti
  4. Valeria Cancila
  5. Claudia Chiodoni
  6. Paola Portararo
  7. Laura Botti
  8. Cesare Valenti
  9. Milena Perrone
  10. Maurilio Ponzoni
  11. Patrizia Comoli
  12. Mara Lecchi
  13. Paolo Verderio
  14. Antonio Curti
  15. Mario P Colombo
  16. Sabina Sangaletti

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    The authors successfully demonstrated the immunogenic effects of a new dendritic cell-based vaccine on AML with NPM1 mutation. Using a transgenic mouse model that carries human NPM1 mutation and an elegant bone marrow chimera approach, they showed that the tumor growth is reduced by the vaccine. Furthermore, they provided evidence for increased CD8+ T cell presence and activity at the tumor site and increased anti-NPMc antibody levels in the serum. These findings are timely and novel and the new methods presented here would be of interest to a broad audience from immunology, inflammation and cancer fields.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Neutrophil extracellular traps (NETs) are web-like chromatin structures composed by dsDNA and histones, decorated with antimicrobial proteins. Their interaction with dendritic cells (DCs) allows DC activation and maturation toward presentation of NET-associated antigens. Differently from other types of cell death that imply protein denaturation, NETosis preserves the proteins localized onto the DNA threads for proper enzymatic activity and conformational status, including immunogenic epitopes. Besides neutrophils, leukemic cells can release extracellular traps displaying leukemia-associated antigens, prototypically mutant nucleophosmin (NPMc+) that upon mutation translocates from nucleolus to the cytoplasm localizing onto NET threads. We tested NPMc+ immunogenicity through a NET/DC vaccine to treat NPMc-driven myeloproliferation in transgenic and transplantable models. Vaccination with DC loaded with NPMc+ NET (NPMc+ NET/DC) reduced myeloproliferation in transgenic mice, favoring the development of antibodies to mutant NPMc and the induction of a CD8 + T-cell response. The efficacy of this vaccine was also tested in mixed NPMc/WT bone marrow (BM) chimeras in a competitive BM transplantation setting, where the NPMc+ NET/DC vaccination impaired the expansion of NPMc+ in favor of WT myeloid compartment. NPMc+ NET/DC vaccination also achieved control of an aggressive leukemia transduced with mutant NPMc, effectively inducing an antileukemia CD8 T-cell memory response.

Article activity feed

  1. Version published to 10.7554/elife.69257 on eLife
  2. eLife

    Author Response:

    Reviewer #1:

    NPM1-mutated acute myeloid leukemia (AML) is a frequent AML subtype for which new therapeutic approaches are needed. Immunotherapy may represent a promising strategy, to be combined with or alternative to chemotherapy. Particularly, vaccination strategies may be successful in NPM1-mutated AML, as mutant NPM1 is known to be immunogenic and specific T-cells may control disease relapse. In this study, Tripodo et al. performed a handful of experiments to demonstrate the feasibility and the antileukemic efficacy of a dendritic cell (DC) vaccine armed with neutrophil extracellular traps (NETs) derived from NPM1-mutated myeloid cells.

    While this work has the major strength of being novel and performed in vivo, the models used in the study, the number of replicates and the quality of some of the data presented seem insufficient.

    One of the major issues is the lack of a formal demonstration of clear antileukemic activity of this approach in leukemic mice. The authors first used a non-leukemic model, then, in their second set of experiments, a subcutaneously injected AML model. In this regard, I am worried that no effect may be seen in AML models where leukemia is engrafted in the bone marrow or in leukemic genetically modified mouse models.

    As for Reviewer suggestion we challenged the possibility of detecting vaccine effect against the leukemia model engrafted into the bone marrow. To this end C1498-NPMc+ also GFP+ cells were injected directly into the tibia of C57BL/6 mice, to better mirror leukemia development and the cross-talk with bone marrow microenvironment. Treating injected mice with NPMc+ NET DCs, we confirmed the efficacy of the vaccination schedule showing a reduced engraftment of C1498 within the BM of treated mice compared with animals left untreated. In the same experiment we compared mice vaccinated with DC+NPMc+NET with those vaccinated with DC+NPMc+peptides. Tumor take was evaluated by FACS as number of GFP+ leukemia cells in flushed BM. We show a reduction in GFP+ cells in the BM of mice that have been vaccinated with both NPMc+ NET/DC and the control DC/peptide vaccine in comparison to non-vaccinated mice (new Figure 4G). However only the reduction obtained through the NPMc+ NET/DC vaccine reached statistical significance (New Figure 4G). Also, the NPMc+ NET/DC vaccine was superior in inducing the proliferation of CD8 T-cells (evaluated as frequency of CD8+Ki-67+, Fig. 4I) and their production of TNF (new Figure 4I). Both vaccines sustained OX40 expression on CD8 T-cells (new Figure 4J) and reduced the frequency of exhausted PD1+TIM3+LAG3+ CD8-T cells (new Figure 4K). -cells (new Figure 4L-M). The production of TNF by CD8 T-cells was higher in NPMc+ NET/DC vaccinated mice compared to controls. In this mice we also observed a trend in the induction of Tem (CD44+CD62L+) (new Figure 4O).

    Finally, both vaccines were able to induce Auto-Ab against mutant NPMc (new Figure 4P), however only NPMc+ NET/DC vaccinated mice developed Ab to MPO (new Figure 4Q). The last might concur to the anti-leukemia response we have particularly observed in the sc setting, indeed C1498 cells do express MPO (Mopin, A 2016).

    Overall, the data support reason and advantage of the newly proposed NET-based vaccines in AML. The use of leukemic blasts as source of NETs provides the cell antigen repertoire displayed onto the DNA threads, also endowed of adjuvant functions. Differently, peptide-based vaccines require prior knowledge of the immunogenic peptides to be loaded into in vitro-activated DCs.

    Furthermore, it is unclear what would be the place of NET-DC among other DC vaccines, as there is no direct comparison in this study.

    We understand the point raised by the reviewer. The requested comparison between NPMc/NET and DC peptides vaccines was already present in the original manuscript (Figure 4C-E and supplementary Figure 2 in the present resubmission) and indicated a more efficient reduction in tumor growth in presence of NET/DC vaccines compared to NPMc+ peptides-based vaccines. Following the Reviewer’s request, we performed additional in vivo experiments comparing the efficacy of the two vaccines in the orthotopic injection setting (intra-bone). Similarities and differences in the two vaccines in terms of leukemia take and CD8 T cell activation status are discussed above and in the manuscript were data are shown as new Figure 4 (panel G-Q).

    Other issues include replicate numbers that need to be increased in some experiments, data representation which is not always appropriate and one panel which has been duplicated from a previously published work.

    To overcome the limitation of the number of observations, we now applied non-parametric approaches in order to have more robust results, according to the new statistical analysis performed.

    In addition for the new experiments we increased the number of observations and results obtained from the new analysis adjusted for the different experiments (block variable) have been reported in the manuscript.

    For all the experiments, we represented individual data as dot plots with the average and appropriate error bars for each group or boxplots. Statistical methods section has been accordingly modified.

    We also thank the Reviewer for pointing out the issue regarding the IF panel, which has been replaced with the correct one.

  3. eLife

    Evaluation Summary:

    The authors successfully demonstrated the immunogenic effects of a new dendritic cell-based vaccine on AML with NPM1 mutation. Using a transgenic mouse model that carries human NPM1 mutation and an elegant bone marrow chimera approach, they showed that the tumor growth is reduced by the vaccine. Furthermore, they provided evidence for increased CD8+ T cell presence and activity at the tumor site and increased anti-NPMc antibody levels in the serum. These findings are timely and novel and the new methods presented here would be of interest to a broad audience from immunology, inflammation and cancer fields.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    NPM1-mutated acute myeloid leukemia (AML) is a frequent AML subtype for which new therapeutic approaches are needed. Immunotherapy may represent a promising strategy, to be combined with or alternative to chemotherapy. Particularly, vaccination strategies may be successful in NPM1-mutated AML, as mutant NPM1 is known to be immunogenic and specific T-cells may control disease relapse. In this study, Tripodo et al. performed a handful of experiments to demonstrate the feasibility and the antileukemic efficacy of a dendritic cell (DC) vaccine armed with neutrophil extracellular traps (NETs) derived from NPM1-mutated myeloid cells.

    While this work has the major strength of being novel and performed in vivo, the models used in the study, the number of replicates and the quality of some of the data presented seem insufficient.

    One of the major issues is the lack of a formal demonstration of clear antileukemic activity of this approach in leukemic mice. The authors first used a non-leukemic model, then, in their second set of experiments, a subcutaneously injected AML model. In this regard, I am worried that no effect may be seen in AML models where leukemia is engrafted in the bone marrow or in leukemic genetically modified mouse models. Furthermore, it is unclear what would be the place of NET-DC among other DC vaccines, as there is no direct comparison in this study.

    Other issues include replicate numbers that need to be increased in some experiments, data representation which is not always appropriate and one panel which has been duplicated from a previously published work.

  5. eLife

    Reviewer #2 (Public Review):

    Here, the authors successfully demonstrated the immunogenic effects of a new dendritic cell-based vaccine on AML with NPM1 mutation. Using a transgenic mouse model that carries human NPM1 mutation and an elegant bone marrow chimera approach, they showed that the tumor growth is reduced by the vaccine. Furthermore, they provided evidence for increased CD8+ T cell presence and activity at the tumor site and increased anti-NPMc antibody levels in the serum. These findings are timely and novel and the new methods presented here would be of interest to a broad audience from immunology, inflammation and cancer fields.

  6. Version published to 10.1101/2021.05.19.444838v1 on bioRxiv