Panel A shows an experimental timeline from day 0 to day 8 covering Treg isolation, lentiviral transduction, phenotyping, and activation assays. Panel B shows flow cytometry plots of CD25 versus CD127 gating from PBMC to CD25 positive to Treg populations at 5.84, 73.1, and 100 percent. Panel C shows a line graph of expansion fold up to 40 over 7 days, where all CAIleR constructs and polyclonal expand similarly. Panel D shows flow cytometry plots of Foxp3 versus Helios with Foxp3 positive Helios positive percentages of 78.1, 85.4, 85.7, 86.4, and 89.4 percent for polyclonal and CAIleR 5, 8, 10, and 11, with a bar graph showing consistent expression around 80 to 90 percent. Panel E shows mCherry versus Bet v1-PE plots confirming transduction with editing efficiencies of 48.16, 51.71, 42, and 59.5 percent for CAIleR 5, 8, 10, and 11, with a bar graph showing all CAIleRs reaching approximately 40 to 60 percent editing efficiency. Panel F shows flow cytometry plots of OX40 versus 41BB after K562 wild type and K562 Bet v1 stimulation, where CAIleR 5 and 8 show the highest activation at 40.2 and 45.6 percent with K562 Bet v1, confirmed by bar graphs with significant differences marked. Panels G and H show CFSE dilution histograms and suppression line graphs for CAIleR 8 Teff and CAIleR 11 Teff targets across Treg to Teff ratios from 1 to 1 to 1 to 64, where CAIleR 5 and 8 Tregs show suppression reaching approximately 80 percent at 1 to 1 ratio, declining gradually at higher dilutions, with significant differences marked across multiple ratios.
In vitro suppression of anti-Bet v1 CAlleR Tregs. (A) Experimental design of human CAlleR Treg production. (B) Representative plots of CD25 and CD127 expression on cells at the different steps of Treg isolation. (C) Expansion of Tregs over 7 days of cell culture. (D) Representative plots and cumulative data of Foxp3 and Helios expression in Tregs after expansion. (E) Representative plots and cumulative data of transduction efficiency of anti-Bet v1 CAlleR Tregs (n = 3–4). (F) Representative plots and cumulative data of CAlleR Treg activation markers (assessed by 41BB and OX40 expression) after 24-h coculture with K562 WT or K562 Bet v1 at a K562:Treg ratio of 1:5. (G and H) Suppressive effect of CAlleR and polyclonal Tregs on (G) CFSE+ CAlleR 8 or (H) CFSE+ CAlleR 11 Teff. Plots show representative CFSE dilutions of Teff after 96-h coculture with K562 Bet v1 (K562 Bet v1:Teff ratio of 1:10) and different ratios of Tregs and cumulative data showing the percentage of proliferation suppression. Data represent the mean ± SEM from three to four independent experiments. Exact P values were determined by an unpaired t test in F and by two-way ANOVA with Dunnett’s test to compare CAlleR with polyclonal Tregs in G and H. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.