Panel A shows an in vivo timeline with Treg injection at day 0, intranasal allergen instillations from day 0 to day 11, and sacrifice at day 14. Panel B shows cells per milliliter in BALF reaching approximately 6 times 10 to the power of 5 for BPE, declining significantly with both CAR A2 and CAIleR 8 Treg treatment. Panel C shows immune cell percentages in BALF where eosinophils reach approximately 60 percent in BPE and decline significantly with CAlleR 8 Tregs, while macrophages show reciprocal increases. Panel D shows IL-10 levels in picograms per milliliter in BALF declining from approximately 150 in PBS to approximately 100 in BPE with significant differences marked for CAIleR 8 Treg. Panel E shows neutrophil, monocyte, and eosinophil percentages gated on CD45. Panel F shows SiglecF dim eosinophils rising and SiglecF high declining significantly with CAIleR 8 Treg treatment. Panel G shows CD4 positive T cell percentages for IL4, IL5, IL13, and IL17 with significant reduced levels of IL4 in CAlleR 8 Treg treated group. Panel H shows hematoxylin/eosin and PAS stained lung histology across PBS, BPE, CAR A2, and CAIleR 8 conditions. Panel I shows PAS positive percent declining from approximately 40 percent in BPE to approximately 25 percent with CAIleR 8 Treg treatment with significant differences marked. Panel J shows line graphs of respiratory mechanics Rn, G, and H in centimeters H2O seconds per centimeter across methacholine concentrations from baseline to 50 milligrams per milliliter, where CAIleR 8 Treg shows significantly lower values at 50 milligrams per milliliter for all three parameters compared to CAR A2 Treg.
CAlleR 8 murine Tregs for the prevention of allergic airway inflammation. (A) In vivo experimental design of BPE-induced allergic airway inflammation. (B) Cell counts in BALF (n = 5–9). (C) Differential counts of cells infiltrating the BALF (n = 5–9). (D) IL-10 quantification in BALF by ELISA. (E) Percentages of granulocytes in the lungs (n = 4–7). (F) SiglecF expression on eosinophils in the lungs (n = 4–7). (G) Th2 cytokine production in CD4+ T cells of the mLN (n = 5–7). (H) Representative images of H/E and PAS staining of the lungs (black bars represent 100-μm scale). (I) Quantification of PAS+ small and medium airways of the lungs (n = 6–9). (J) FlexiVent measurement of Newtonian resistance (Rn), tissue elastance (H), and tissue damping (G) at baseline and at different doses of inhaled methacholine (n = 5). Data are presented as the mean ± SEM from two independent experiments (except for J that shows data from one experiment). P values were determined by one-way ANOVA with Tukey’s multiple comparison test in B, C, D, E, G, and I and with two-way ANOVA with Tukey’s multiple comparison test in F and Sidak’s test in J. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. H/E, hematoxylin/eosin.