Figure 2. Thymocyte Apoptosis in Response to a Defined Number of Peptide-MHC Ligands.

CH27 cells were loaded with biotin-MCC, extensively washed and labeled with SA-PE, and imaged together with 5c.c7 Cd74^−/− thymocytes in the presence of 2 mM Ca²⁺ and Annexin V-FITC. SA-PE signal was acquired within 5 min of interaction in 1 micron sections and then reconstructed with Metamorph software so that the number of SA-PE molecules present could be determined. FITC fluorescence was assessed every 15 min for 6.5 hr.

(A and B) A DIC image (A) and PE fluorescence image (B) of a thymocyte-CH27 interaction. PE fluorescence images are rotated 90° so that the thymocyte-APC interface faces the viewer. This interface contains a single peak of PE fluorescence corresponding in fluorescence intensity to a single SA-PE molecule.

(C) DIC (top) and Annexin V-FITC fluorescence images (bottom) of this cell couple over time. Frames are taken 30 min apart; the last frame represents 6 hr after interaction.

(D and E) A DIC image (D) and PE fluorescence image (E) of a thymocyte-CH27 interaction in which the interface contains a single peak of PE fluorescence with double the intensity of that in (B), corresponding to 2 SA-PE molecules.

(F) A time-lapse of DIC and Annexin V-FITC shows that the thymocyte has committed to apoptosis after 1.5–2 hr of interaction (frames are 30 min).

(G) Summary of data represented in (A)–(F), n = 12–18 cell couples for each group.