Extended Data Fig. 1: Shorter exposure to TIFM has effect on T cell inhibitory receptor function and Granzyme B expression.
From: Tumour interstitial fluid-enriched phosphoethanolamine suppresses T cell function
A) %PD1hi cells from Fig. 1c. Data are ±SEM from four independent experiments (n = 8 mice/group (p-value 0.0018). B-E) Cells from Fig. 1d-f analyzed for B) Normalized IFNγ MFI±SEM C) Normalized TNF MFI ±SEM D) %Granzyme Bhi cells and E) %IL-2hi cells. Data quantified from four independent experiments (n = 8-9 mice/group, B) p = 0.1918, C) p = 0.3385 D) p < 0.0001 E) p = 0.5219). Statistics were calculated using a two-tailed paired t-test. B) p-value F) Timeline Schematic of experiments in G-J G) Normalized PD-1 MFI (mean fluorescence intensity) ±SEM from four independent experiments (n = 8-9 mice/group, p-value RPMI vs. T3 p = 0.0010, T1 vs. T3 p = 0.0061). H) Day 7 OT-I CD8+ T cells were analyzed for IFNγ+TNF+ co-expression after 6 hours αCD3/αCD28 restimulation. Data quantified show %IFNγ+TNF+ events among Live CD8+ T cells ±SEM from four independent experiments (n = 8 mice/group). I-J) Day 7 OT-I T cells were analyzed for expression of I) IL-2 or J) Granzyme B after 6 hours of αCD3/αCD28 restimulation. Data quantified show normalized MFI ±SEM from four independent experiments (n = 8-9 mice/group, J) p-value RPMI vs. T3 p < 0.0001, RPMI vs. T3, p = 0.0026). K) Day 7 OT-I CD8+ T cells were co-cultured with B16-OVA cells at a 1:1 effector:target ratio in an Agilent xCELLigence RTCA DP system to monitor target cell killing. XY plot on the left shows %Cytolysis ±SEM, and Area under the curve values are quantified from three independent experiments (n = 5-6 mice/group, p values RPMI vs. T1 p = 0.9897, RPMI vs. T3, p = 0.0179, T1 vs. T3, p = 0.0224). Statistical analysis for all indicated comparisons in F-K) was determined using a one-way ANOVA with Tukey’s multiple comparisons test. L-O) Cells from Fig. 1h-j were restimulated with αCD3/αCD28 in RPMI or TIFM for 6 hours. After restimulation, cells were analyzed for L) Normalized IFNγ MFI of Live IFN+ CD8+ Cells, M) Normalized TNF MFI of Live TNF+CD8+ cells N) % of Granzyme Bhi cells and J) % of IL-2hi cells. (n = 8 mice/group from three independent experiments). For L-O), statistics were calculated using a two-tailed paired t-test (p-values L) p = 0.0268 M) p < 0.0001, N) p < 0.0001, O) p = 0.0006) Source numerical data are available in source data.
