Panel A shows CAR A2 and CAIleR 8 construct schematics with GFP reporter and a timeline from day 0 to day 7 covering Treg isolation, retroviral transduction, and intravenous injection. Panel B shows flow cytometry plots confirming CD25 positive Foxp3 positive at 87.5 and 89.3 percent for CAR A2 and CAIleR 8 Tregs, with GFP positive at 90.1 percent for CAR A2 and with GFP/Bet v1 positive at 47.9 percent for CAlleR 8. Panel C shows an experimental timeline from day 0 to day 5 with intranasal allergen instillations, Treg injection at day 0, and sacrifice with organ embedding at day 5. Panel D shows multiplex immunofluorescence images of mediastinal lymph node, lung, and spleen across PBS CAIleR 8, BPE CAR A2, and BPE CAIleR 8 conditions, stained with DAPI, GFP, CD11c, and B220, with inset panels showing individual channel detail. Panel E shows scatter dot plots of GFP positive cells per square millimeter in mediastinal lymph node, lung, and spleen, where BPE CAIleR 8 shows significantly higher cell density reaching approximately 20 to 25 cells per square millimeter in mediastinal lymph node and approximately 1.5 cells per square millimeter in lung compared to other conditions. Panel F shows scatter dot plots of percent of GFP positive cells interacting with CD11c positive, B220 positive, and no contact populations across mediastinal lymph node, lung, and spleen, where BPE CAIleR 8 shows significantly higher CD11c positive interactions in mediastinal lymph node reaching approximately 40 percent, while lung shows predominantly no contact interactions above 80 percent for BPE conditions with significant differences marked.
CAlleR 8 murine Treg tissue–specific homing. (A) Experimental design of murine CAlleR 8 GFP+ and CAR A2 GFP+ Treg production. (B) Representative plots of Treg phenotype and transduction efficiency after 7 days of expansion. (C) Experimental design of the allergic airway inflammation mouse model and adoptive Treg transfer. (D) Representative images of CAlleR 8 and CAR A2 Tregs in the mLNs, lung, and spleen of mice exposed to PBS or BPE. White bars represent the scale of 20 μm, and small images show fluorescence channels of the magnified region outlined in white squares. (E) Cumulative data of detection of GFP+ cells/mm2 in each organ (n = 8 in the lung and spleen and n = 5–8 in mLN from four mice with image duplicates). (F) Cumulative data of the percentage of GFP+ cells interacting with CD11c+, B220+ APCs or with none of them (no CD11c+/B220+ contact) for each organ (n = 8 in the lung and spleen and n = 5–8 in the mLN from four mice with image duplicates). In E, data are represented by the median ± interquartile range and in F by the mean ± SEM. P values were calculated with Kruskal–Wallis and Dunn’s multiple comparison test in E and with two-way ANOVA with Tukey’s test in F. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.