Genetic reconstitution with wild-type AP3D1 restores expression and function of the AP3 complex. (A) Immunofluorescence analysis of patient PHA/IL-2 blasts reconstituted with either empty vector or wild-type AP3D1 using mouse anti-AP3δ (SA4). Cells are shown in one 3D stack picture of 28 slices. Cells successfully transduced with the vector are GFP-positive (white in the single stains and green in the merged picture), the nucleus is stained with Hoechst (blue), and AP3δ is stained with SA4 provided by A. Peden¹⁸  (white in the single stains and red in the merged picture). The variable GFP intensity of reconstituted cells is explained by the use of an IRES-GFP construct in which AP3D1 is placed before and GFP is placed after the IRES sequence. (B) Transduced T cells were rested in medium free of IL-2 for 24 hours and then incubated in medium or stimulated with 3 µg/mL plate-bound CD3 for 3 hours. Transduction efficiencies varied between 1% and 12%. (C) CD107a expression after incubation of transduced cells with medium or plate-bound anti-CD3. Plots are gated on successfully transduced GFP-positive CD8⁺ cells. The solid line indicates peak CD107a expression in patient cells transduced with the empty vector. (D) Overlay of CD107a expression of transduced T cells kept in medium (dotted line) and (E) stimulated with anti-CD3 (solid line). The difference in mean fluorescence intensity (ΔMFI) of CD107a expression between cells incubated in medium vs stimulated with plate-bound anti-CD3 is shown for patient (Pat) and control (ctr) GFP-positive CD8⁺ T cells transduced with the indicated vector. Results are shown from 2 independent experiments.