Figure 3.

CD8+ OT-1 lymphocytes are activated by P2X7-dependent cross-dressing

(A) ACs generated from EL4 cells (E-ACs) by nutrient deprivation-induced cell death were identified with Annexin V (AV) and Propidium Iodide (PI) on the seventh day of culture. Representative dot plots of flow cytometry analysis show two populations of E-ACs: late Apoptotic (PI + AV+) and early apoptotic (PI-AV+) E-ACs. Data represents one of three independent experiments. Only PI-AV- cells are present at the beginning of treatment.

(B) Western blot for P2X7 receptor from EL4 live cells, E-ACs, EL4 P2X7 live cells, and E-ACs-P2X7, 1–4 lanes, left to right, respectively. The 75 kDa band corresponds to the molecular weight of the P2X7 receptor.

(C) The SIINFEKL/H2-K^b presence was evaluated by flow cytometry in ACs generated from both parental (E-ACs, left side) and P2X7 overexpressing (E-ACs-P2X7, right side) H-2K^b EL4 cells. SIINFEKL/H2-K^b in unloaded ACs (blue) and peptide-loaded ACs (red) were compared against basal fluorescence (no antibody, black).

(D) Evaluation of antigen/MHC (SIINFEKL/H2-K^b complex) transference from ACs to the surface of allogenic (H-2^d) BMDCs-BALB/c. A representative histogram from 15 independent experiments of the SIINFEKL/H2-K^b detected in CD11+ BM-DCs challenged with E-ACs (gray), E-ACs-P2X7 (pink), E-ACs SIINFEKL (blue), and E-ACs-P2X7/SIINFEKL (red). As controls, BMDCs-BALB/c not challenged (black) and challenged with SIINFEKL peptide (green) were included.

(E) The quantification of at least six independent experiments was graphed and represented as the mean ± SEM, SIINFEKL/H2-K^b MFI in CD11⁺ BM-DCs normalized to DCs not challenged. Gray circles represent BMDCs-BALB/c pretreated with oATP before challenge with each treatment. Data are represented as mean ± SEM. Results were analyzed using non-parametric Mann-Whitney test, ∗∗ p < 0.01.