Fig 1: Assessment of the functional impact of neutrophils from Ax-3 crew on T cell responses(A) CD3+ T cells isolated from the peripheral blood of the astronauts before and after the mission were stimulated with anti-CD3/28 beads and proliferation responses were measured by a CFSE-based flow cytometric assay. Histograms obtained from an astronaut are shown as a representative result. T cells from healthy volunteers (ground controls) were also studied (gray histogram).(B) A schematic (created in BioRender Created in BioRender. Esendagli, G. (2025) https://BioRender.com/a4m1kdr) is presented for the experimental setup used for testing the functional impact of neutrophils. NDN and LDN cells collected from astronauts before (L-1d) and after (R+1day) the Ax-3 mission were co-cultured with the anti-CD3/CD28-stimulated T cells obtained from healthy control individuals. T cells proliferation (C), and secretion of IL-2 (D) and IFN-γ (E) were determined following incubation for 72 h. Statistical analyses were performed with paired t test (∗∗p ≤ 0.01). The p values calculated to be > 0.05 were not indicated. The data are presented as mean with standard error of mean (SEM).
Fig 2: Comparison of the impact of space flight and suborbital flight on immunophenotypic and functional characters of NDN(A) Granularity (SSC) and size (FSC) values of NDN before and after the Ax-3 mission and the suborbital flight mission were quantified by flow cytometry.(B–D) Expression levels of the neutrophil surface molecules were calculated by normalizing the median fluorescence intensity (MFI) to AF for each marker and a comparative heatmap was prepared (A.U., arbitrary units). The change in ROS (C) and NO (D) production capacities in NDN after the Ax-3 mission and the suborbital flight was calculated by comparing the data of the NDN obtained before the mission.(E–H) NDN cells collected from the astronauts before and after the Ax-3 and suborbital flight missions were co-cultured with the anti-CD3/CD28-stimulated T cells from healthy control individuals. T cell proliferation in the co-cultures with increasing amounts of NDN was normalized to that of the T cells stimulated alone. In addition, the changes in T cell proliferation (F), and secretion of IL-2 (G) and IFN-γ (H) in the presence of NDN after the Ax-3 mission and the suborbital flight were calculated by comparing to the data obtained with the NDN collected before the missions. Statistical analyses were performed with paired t test (∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001). The p values calculated to be > 0.05 were not indicated. The data are presented as mean with standard error of mean (SEM).
Fig 3: Human TriStim-E6/E7 mRNA did not induced cytokine release syndrome in an immune system humanized mouse model. (A) M-NSG mice were engrafted with human PBMCs. Twenty days post-engraftment, reconstitution of human leukocytes was confirmed by flow cytometry analysis of mouse peripheral blood samples using an anti-human CD45 antibody. (B) On day 21, PBMC-reconstituted mice were intramuscularly vaccinated with 20 μg of hTriStim-E6/E7 mRNA-LNP or control empty, or intravenously injected with 2 mg/kg of TGN1412 and their serum cytokines (IFNγ, IL-10, IL-6, IL-2, IL-4, and TNFα) were determined by cytometric bead array analysis 6 and 24 h post-treatment. Data are presented as the mean ± standard deviation (n = 5). Data are representative of two independent experiments.
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