FIGURE 3 from p35 is a Crucial Player in NK-cell Cytotoxicity and TGFβ-mediated NK-cell Dysfunction
Year: 2023
Type: other
Abstract: Murine NK cells from p35 k/o mice display heightened cytotoxicity. A, Murine splenocytes or bone marrow cells from WT or p35 k/o mice were stained for CD3 and NK1.1 and mixed with counting beads, then analyzed via flow cytometry. Gating on NK1.1+CD3− cells, as well as the counting beads, allowed determination of mNK concentration and the total mNK cell count in each compartment. ns, not significant. Graphs display mean ± SD, n = 3 mice, unpaired two-tailed t test. B, mNK cells were isolated from WT, p35 k/o, or p35 Het mice and cocultured with calcein AM-labeled B16F10 cells at different E:T ratios for 4 hours. Fluorescence intensity of supernatant corresponding to calcein release from dead cells was measured to calculate NK-mediated cytotoxicity. **, P < 0.01. Graphs display mean ± SD, n = 3 biological cocultures (n = 2 for WT 1:1 ratio, p35 k/o 1:1 ratio, and p35 k/o in WT versus p35 Het versus p35 k/o experiment), and multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method (WT vs. p35 k/o) or one-way ANOVA with Tukey multiple comparisons test (WT vs. p35 Het vs. p35 k/o). C, Murine splenocytes or bone marrow cells were stained for CD11b, CD27, CD3, and NK1.1 and analyzed via flow cytometry after gating on NK1.1+CD3− cells. Representative flow plots are shown. Percentages of mNK cells in each subset (CD11b+CD27−, CD11b−CD27+, and CD11b+CD27+) for each mouse were recorded and graphed. ns, not significant. Graphs display mean ± SD, n = 8 mice, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. D, Murine splenocytes were stained for CD3, NK1.1, and NKG2D, NKp46, NKG2A, 2B4, LY6C, or LY49C/F/I/H. Representative histograms of activating/inhibitory receptor expression are shown, gated on live NK1.1+CD3− cells. E, Isolated mNK cells pooled from multiple mice were seeded at a concentration of 2e6 cells/mL and cultured ex vivo in RPMI media supplemented with 1,000 U/mL IL2. An additional 1,000 U/mL IL2 was added 2 days later without changing the media, followed by collection of supernatant after culture for an additional 2 days. Cytokine concentration was determined using a flow-based, multiplex murine cytokine release assay. ns, not significant; *, P < 0.05; **, P < 0.01. Graphs display mean ± SD, n = 3 biological cultures, unpaired two-tailed t tests.
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4372286239