Generation of HLA-A*02-specific CAR CD8⁺ Tregs. (A) Gating strategy for CD8⁺CD45RC^low nTreg sorting by FACS Aria from PBMCs. CD8⁺ Tregs were selected on morphology, exclusion of doublets and dead cells, and positive selection of CD8⁺ and CD45RC^−/low expression. (B) Total T cells and purified CD8⁺ Tregs were stimulated ON with anti-CD3 and anti-CD28 mAbs (day 0), transduced with CAR recombinant lentivirus at days 1 and 2 in a range of multiplicity of infection from 2 to 10, calculated on lentivirus titration on Jurkat cells. CAR expression was analyzed at day 8 by flow cytometry staining of cMyc and LNGFR. (C) Schematic depicting the process for CAR CD8⁺ Treg production. CD8⁺ Tregs were isolated, stimulated, transduced twice with CAR-encoding lentivirus, expanded, selected on CAR expression, and expanded for a total culture of 14 days. (D) FACS analysis of CAR expression by CD8⁺ Tregs after 7 days of culture based on LNGFR staining. (Left) Percentage of cells expressing the A2-CAR (n = 24) or Her2-CAR (n = 22) at day 7 before purification. Mean ± standard error of the mean (SEM) is shown. (Right) Representative histograms of LNGFR expression in A2- and Her2-CAR transduced CD8⁺ Tregs compared with nontransduced cells. (E) Representative histogram of LNFGR staining on CD8⁺ Tregs cultured 14 days previously purified at day 7 on A2- or Her2-CAR expression compared with NT cells. (F) Expansion fold of CD8⁺ Tregs from day 0 plating to day 14 harvesting when transduced with A2-CAR (n = 19) or Her2-CAR (n = 18) compared with NT (n = 4). Mean ± SEM. (G) A2-CAR and Her2-CAR CD8⁺ Tregs were incubated or not (unstained) with HLA-A*02 tetramer labeled with PE and assessed for anti-HLA-A*02 recognition. Representative histogram of 3 experiments.