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. 2022 Jul 6;13:931630. doi: 10.3389/fimmu.2022.931630

Figure 4.

Figure 4

NKG7 shortens immune synapse duration and promotes efficient serial killing. (A) Frequency of CD8+ T cells conjugated to P815 target cells at indicated timepoints evaluated by flow cytometry. 72 hour activated Nkg7+/+ or Nkg7-/- CD8+ T cells were labelled with cell trace violet (CTV) and co-cultured with anti-CD3/28-coated GFP-labelled P815 cells. Time indicates minutes of co-culture. Plots are gated on CTV+ cells and frequency of conjugates was calculated as CTV+GFP+ as a percent of all CTV+ cells. Data is quantified in right panel, Wilcoxin matched-pairs signed rank test, n = 3. (B) Live cell imaging time-lapse montage of CTV-labelled 72 hour activated Nkg7+/+ or Nkg7-/- CD8+ T cells (blue) co-cultured with anti-CD3/28 antibody-coated P815 target cells. Lysotracker (green) was added to co-cultures for cell clarity and PI was added to measure target cell death. Time indicates hours of co-culture. (C) From (B), time taken between T cell/target conjugation and target cell death as measured by PI uptake, unpaired t test with Welch’s correction, n = 21-55 T cells evaluated, pooled from 2 independent experiments over a 14-hour co-culture. (D) Frequency of T cells from (B) making no contact with any target (No contact), making contact without killing (Contact no kill), killing a single target only (Single kill) or killing more than one target (Serial kill) during the first 7 hours of co-culture. (E) Specific lysis of 51Cr-labelled anti-CD3/28-coated P815 tumor cells (targets) co-cultured with 72 hour activated Nkg7+/+ or Nkg7-/- CD8+ T cells (effectors) at an effector to target ratio of 1:5 for indicated lengths of time, measured by 51Cr release. Data was normalized by converting the maximum killing by Nkg7+/+ CD8+ T cells to 100%. (F) Relative rate of killing measured from assays set up as in (E) at indicated effector to target ratios, with relative rate measured as the slope of the Nkg7-/- killing curve relative to the Nkg7+/+ killing curve at the corresponding effector to target ratio, 2-way ANOVA Sidak’s multiple comparisons test, n = 3. All error bars show +/- SEM. ns – not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.