doi: 10.1038/s41375-019-0585-7.
Epub 2019 Oct 4.
PMN-MDSC are a new target to rescue graft-versus-leukemia activity of NK cells in haplo-HSC transplantation
Affiliations
- PMID: 31586150
- PMCID: PMC7214239
- DOI: 10.1038/s41375-019-0585-7
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PMN-MDSC are a new target to rescue graft-versus-leukemia activity of NK cells in haplo-HSC transplantation
Leukemia.
2020 Mar.
No abstract available
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Presence of PMN-MDSC in the apheresis of G-CSF-mobilized donors and their effect on NK/ILC differentiation from HSC. a–c Mononuclear cells present in the apheresis were analyzed by flow-cytometry for the expression of specific markers allowing the identification of MDSC subsets. a Gating strategy adopted in one representative experiment out of 70 performed. b Percentages and c absolute numbers of PMN-MDSC (CD66b+ cells) in different donors before (pre) and after (post) mobilization (mob). d Percentages of PMN-MDSC viable cells at different time points. e–g In these experiments, CD34+ HSC were isolated from the apheresis of G-CSF-mobilized donors and cultured with cytokine-medium either in the absence (ctr) or in the presence (+MDSC) of PMN-MDSC (at 1/1 ratio) and analyzed by flow-cytometry for: e percentages ± SEM of CD56+ cells at different time points (n = 7); f RORγt and Eomes transcription factor expression in gated CD56+CD94+ cells (NK cells) and CD56+CD94− (ILC3) at day 40 of culture. One representative experiment out of seven performed; g expression of informative intracellular cytokines (IFN-γ, IL-22, TNF-α, and IL-8) at day 40 in CD56+CD94+ or CD56+CD94− cells after stimulation with PMA+Ionomycine+IL-23. Percentage ± SEM (n = 3–7). A p value ≤ 0.05 was considered statistically significant. *p ≤ 0 .05; **p ≤ 0 .01; *** p ≤ 0.001; **** p ≤ 0.0001; ns = not significant. Where not indicated, the data were not statistically significant
PMN-MDSC inhibit NK-cell function via IDO, PGE2, and exosomes. a–d In these experiments, the acute lymphoblastic leukemia cell line NALM-18 was used as target to asses either the NK-cell cytolytic activity or their cytokine production. Freshly isolated or IL-2-expanded NK cells were cultured either alone (NK) or in the presence of PMN-MDSC at 1/1 ratio. After 48 h of co-culture, PMN-MDSC were removed by magnetic cell separation. The resulting NK cells were used as effector cells (indicated as NK/MDSC) in the cytolytic, cytokine and degranulation assays. Percentages of killed target cells by a freshly isolated (n = 4) or b IL-2-cultured NK cells (n = 6). b NK cells were cultured either alone or under cell-to-cell contact (NK/MDSC) or under transwell (Tw) conditions as indicated in the figure. Cytokine production and degranulation of c freshly isolated (n = 3) or d IL-2 expanded NK cells (n = 5). Bars indicate the PMN-MDSC-mediated inhibition (% ± SEM), referred as the ratio between NK/MDSC co-cultures and NK cells cultured alone (arbitrarily normalized to 100). e–g Evaluation of the effect of donor PMN-MDSC on autologous NK-cell cytotoxicity and degranulation capability (CD107a) against recipient leukemia blasts. e Percentages of killed patient’s blasts by freshly isolated donor NK cells (n = 5). f One representative contour plots and g percentages ± SEM of CD107a expression against patient’s leukemia blasts (n = 3). In h–i were analyzed the mechanism(s) involved in the NK-cell inhibition. IL-2-expanded NK cells were cultured either alone (ctr., black bars) or in the presence of PMN-MDSC at 1/1 ratio. After 48 h of co-culture, PMN-MDSC were removed by magnetic cell separation. The resulting NK cells (referred as +MDSC, gray bars) were analyzed. Mean fluorescence intensity (MFI) ± SEM of the indicated (h) surface markers (n = 12), (i) granzyme B (n = 8), perforin (n = 9) and signaling molecules CD3ζ (n = 9), and DAP12 (n = 6). j IL-2-expanded NK cells were cultured either alone or with PMN-MDSC (at 1/1 ratio) in the absence or in the presence of 1-MT (IDO inhibitor) and/or NS398 (PGE2 inhibitor). The cytolytic activity of NK cells cultured under different conditions was tested against NALM-18 cells (n = 6). Percentages ± SEM of killed cells (E/T ratio used was 10/1). k Western blot analysis of calnexin, CD63, and TSG101 in PMN-MDSC cell lysates and in PMN-MDSC-derived exosome. l–o Confocal microscopy of NK cells incubated for 48 h with exosomes purified from PMN-MDSC (5 µg/5 × 105 cells). Confocal microscopic analysis of l CD56+ NK cells (red), m PKH67+-exosomes (green), n merge of CD56+ and exosome, nuclei in DAPI (blue); o confocal z-stack of merged section. Magnification ×60. o, p Percentages of killed NALM-18 cells after incubation with PMN-MDSC-derived exosomes. p One representative experiment out of six performed and statistical analysis. A p value ≤ 0.05 was considered statistically significant. *p ≤ 0 .05; **p ≤ 0 .01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = not significant. Where not indicated, the data were not statistically significant
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