Figure 3. Phospholipid reactivity of T cells binding lysylPG-treated CD1a tetramers.
(a) Phospholipid structures: phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylglycerol (PG), and lysylphosphatidylglycerol (lysylPG). (b) Flowcytometric analysis of CD1a-lysylPG reactive T cell line (921a) stained with CD1a tetramers loaded with indicated phospholipids. Gated for live cells. Results were representative of >3 independent experiments. (c) Overlapping histograms of flowcytometric analysis of CD1a-lysylPG reactive T cell clones (2114.1, 966.1.4-) stained with CD1a tetramers loaded with indicated phospholipids. (d) Surface plasmon resonance was used to measure the affinity of the interaction between soluble 921.3 TCR and immobilized untreated CD1a (CD1a-endo, red) and PG (green) or lysylPG (blue)- treated CD1a. Shown binding curves correspond to one experiment with two injections for each concentration of the TCR (from 0 to 150 μM). Equilibrium KD was calculated from two independent measurements. (e) Flowcytometric analysis of T cell lines from donors 834 and 966 co-stained with CD1a-lysylPG PE-labeled tetramers and CD1a-PG APC-labeled tetramers. Cells were gated for live CD4+ T cells, and distinct populations of tetramer+ T cells are indicated. (f) Flowcytometric analysis of PBMC stained ex vivo with CD1a-lysylPG PE-labeled tetramers and CD1a-PG APC-labeled tetramers. Cells were gated for live CD14-/CD19-/CD3+/CD4+ T cells according to the gating strategy in Supplementary Figure 1a. Percentages of single and double tetramer staining cells are indicated in the quadrants.