(A) Transwell migration of ex vivo expanded TILs from two melanoma patients stimulated with LPA(18:1) at the indicated concentrations. Chemokine CXCL10 (1 μM) was used as positive control; “control” refers to serum-free medium. Agonists were added to the bottom wells and incubation was carried out for 2 h at 37°C.
(B) LPA dose-dependency of migration. The inset shows a representative transwell filter after staining. Migration was quantified by color intensity using ImageJ.
(C) LPA overrules CXCL10-induced TIL chemotaxis. LPA(18:1) was added together with CXCL10 at the indicated concentrations.
(D) Migration of CD8+ T cells isolated from peripheral blood, measured in the absence (control) and presence of the indicated concentrations of LPA(18:1). Note that the presence of 0.5% serum has no effect.
(E) Recombinant ATX (20 nM) added together with the indicated concentrations of LPC(18:1) recapitulates the inhibitory effects of LPA(18:1) on TILs and CD8+ T cells.
(A and C–E) Results are representative of three independent experiments each performed in technical triplicates and expressed as means ± SEM; bars annotated with different letters were significantly different according to Fisher’s least significant difference (LSD) test (p ≤ 0.05) after ANOVA.