Supplementary Information
Figure Legends
Fig.S1:Tcell subsets in AnxA6/and WT mice.
Lymph nodes from untreated AnxA6-/- and WT mice were stained for CD4, CD8 (A-2), CD62L (A-3 and A-4) and CD44 (A-3 and A-4) and analysed by flow cytometry. Graphs in B-D show levels of lymphocytes, CD4+ and CD8+ detected in lymph nodes of wild type to AnxA6/ mice. The presence of naïve (CD62L+CD44-), effector (CD62L-CD44+) and memory Tcells (CD62L+CD44+) was analysed for CD4+ (C) and CD8+ Tcells (D). In B-D, data and error bars are mean and standard deviations, respectively, from three independent experiments. ns, not significant, p>0.05 (2-way ANOVA).
Fig.S2: Thickness of ear pinna and lymph node weight.
(A) The thickness of the ear pinna thickness was measured with a micrometre 24 hours after exposure to DNFB. Left ears (filled bars) of control (Control) WT (dark grey) and AnxA6-/- (light grey) mice and mice previously sensitised to DNFB (Sensitised (DNFB)) were painted with the vehicle alone; right ears (dashed bars) with 0.2% DNFB to elicit an immune response. Data and error bars are mean and standard deviations, respectively, from five independent experiments. ****p<0.0001 (2-way ANOVA). (B) Inguinal lymph nodes were weighed and their weight was expressed as the relative percentage of their body weight. Relative weight of lymph nodes of the control group is shown in plain dark grey (WT) and light grey(AnxA6-/-) bars, and in dashed dark grey (WT) and light grey(AnxA6-/-) bars for the mice previously sensitised to DNFB. Data and error bars are mean and standard deviations, respectively, from five independent experiments. **p<0.01 (1-way ANOVA).
Fig. S3: Characterisation of Tcells isolated from spleen used in ex vivo experiments.
Tcells were isolated from a single cell suspension derived from spleens by depleting non-Tcells. To assess the purity of isolated cells, Tcells were labelled with anti-CD3-PE-Cy7, anti-CD4-V500 and anti-CD8-PacificBlue and analysed by flow cytometry. (A) Lymphocyte population was selected based on the position in forward scatter (FSC)/sideward scatter (SSC) plot. (B) The CD3+ population was selected according to fluorescence intensity of the PE-Cy7 signal. (C) Overlay of an IgG-PE-Cy7 isotope control (gray) with a sample (black). Analysis showed that ~90% of isolated Tcells within the lymphocytes gate were CD3+ Tcells. (D) CD4+ and CD8+ Tcell populations were selected from CD3+ cells by plotting PacificBlue fluorescence versus V500 fluorescence. Typically, ~64% of the CD3+ Tcells were CD4+ and ~30% were CD8+ Tcells. (E) Overlay of an IgG-V500 and IgG-PacificBlue isotype control (gray) with a sample (black). (F) CD3-CD4+ and CD3-CD8+ cells shows that the CD3- population largely consisted of CD4-CD8- cells. Plots in A-B, D and F represent the density of detected signals (gray to black from high to low). Percentages shown in plots refer to the respective parent population with exception of (F) which shows percentage of CD3- subsets within lymphocytes. Plots and percentages are one replicate of a flow cytometry analysis performed in triplicate.
Fig. S4: Analysis of the distribution of phospholipid classes in lysates of primary Tcells from WT (dark grey) and AnxA6/ (light grey) mice.
Quantified amounts of total lipids of one class were expressed as percentages of the total molar amount of phospholipids(PC-O, PC ether lipids; PE-O, PE ether lipids). Data are mean and standard deviations of measurements performed in triplicates with a pooled sample from two mice per group. *p<0.05 (2-way ANOVA).
Figures
Fig.S1:Tcell subsets in AnxA6/and WT mice.
Fig. S2.Thickness of ear pinna and lymph node weight.
Fig. S3: Characterisation of Tcells isolated from spleen used in ex vivo experiments.
Fig. S4: Analysis of the distribution of phospholipid classes in lysates of primary Tcells from WT (dark grey) andAnxA6/ (light grey) mice.