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. 2014 Dec 22;112(1):202–207. doi: 10.1073/pnas.1418690112

Fig. 5.

Fig. 5.

Dipeptides catalyze peptide exchange on the cell-surface class I molecules. (A) Exchange of Kb-bound FAPGNYPAL peptide on the surface of RMA-S cells. RMA-S cells were incubated overnight at 25 °C with 10 µM FAPGNYPAL or SIINFEKFITCL (as a positive control, dashed black line). Then excess peptide was washed off, and 1 µM SIINFEKFITCL was added in the presence of 10 mM GA or GCha or without dipeptide. Binding of SIINFEKFITCL was detected by flow cytometry; the shadow curve is the cell autofluorescence background. (B) An analogous experiment; instead of SIINFEKFITCL, SIINFEKL (10 µM) was used. Binding of SIINFEKL was detected by flow cytometry using the Kb-SIINFEKL–specific monoclonal antibody 25D1.16; the shadow curve is the control (no primary antibody). (C) Exchange of Kb-bound endogenous peptides on the surface of RMA cells. Cells were incubated with 5 µM SIINFEKFITCL in the presence of 10 mM GCha or without any dipeptide. Binding of SIINFEKFITCL was detected by flow cytometry; the shadow curve is the control without SIINFEKFITCL. (D) Exchange of A2-bound endogenous peptide on the surface of T1 cells. Cells were incubated with the A2-specific peptide NLVPKFITCVATV (5 µM) in the absence or presence of 10 mM GA or GM. Binding of NLVPKFITCVATV was detected by flow cytometry; the shadow curve is the control without NLVPKFITCVATV. Data in A–D are representative of at least three independent experiments. (E) Activation of the B3Z hybridoma after cell-surface exchange of Kb–FAPGNYPAL for SIINFEKL on RMA-S cells (with or without the indicated dipeptides). After the exchange reaction, RMA-S cells were incubated with B3Z cells overnight at 37 °C. On the next day, activation of B3Z was detected by a β-galactosidase assay. Data (average of three independent experiments ± SD) were normalized to the positive control (SIINFEKL added to RMA-S cells after overnight incubation at 25 °C). (F) Activation of B3Z hybridoma after peptide exchange on the surface of RMA cells. Data shown are the average of three independent experiments ± SD.