Figure 1.

Generation of different human Treg types. (A) Cartoon of lentiviral expression constructs used in this study. Indicated elements are as follows: interleukin‐10 (IL‐10); CAR specific to the human HLA‐A2 antigen (HLA‐A2‐CAR); radionuclide‐fluorescence reporter formed of the sodium iodide symporter (NIS) fused to the red fluorescent protein TagRFP (NIS‐TRFP). All constructs were joined with T2A cleavage self‐cleavage sites and the cassette was driven by the spleen foci‐forming virus promoter (P_SFFV). The total ORF lengths were 4.8, 4.2, 3, and 2.7 kb, respectively. Required regions in the lentiviral backbone including the P_SFFV promotor added 3.8 kb to each construct to form the complete gene transfer cassette. (B) Percentage of TRFP‐positive Tregs upon harvest at day 20 and measured by flow cytometry. (C) Representative immunoblot of indicated Tregs. Expected patterns for glycosylated and nonglycosylated NIS‐RFP were observed in transduced cells with GAPDH as a loading control. Representative data from the one of three experiments (same donor) are shown. See Supporting Information Fig. S11 for uncropped immunoblot. (D) IL‐10 analysis of Treg culture supernatant on day 20 by ELISA. (E) HLA‐A2‐specific and HLA‐B7 ("irrelevant") dextramers were used to quantify CAR surface expression on day 20 by flow cytometry. Data show means of n = 6 (B, E) or n = 3 (D) donors, with one donor per experiment; error bars are mean ± SEM. p‐Values calculated by comparing A2 and B7 conditions for each cell type using an unpaired Student's t‐test.