Panel A shows construct schematics for CAR A2 and CAIleR 8 Tregs with Thy1.1, P2A, signal peptide, scFv, CD28 domains, and CD3 zeta, alongside a timeline from day 0 to day 7 covering Treg isolation, retroviral transduction, and phenotyping. Panel B shows flow cytometry plots confirming Foxp3 positive CD25 positive at 93.9 and 90.8 percent, Thy1.1 positive Bet v1 positive at 69.7 and 40.4 percent for CAR A2 and CAIleR 8 Tregs, with bar graphs showing consistent Foxp3 and Thy1.1 expression and a line graph showing expansion fold reaching approximately 10 to 12 by day 7. Panel C shows an in vivo timeline with intranasal allergen instillations from day 0 to day 11, Treg injection at day 8, and sacrifice at day 15. Panel D shows a scatter dot plot of cells per milliliter in BALF reaching approximately 1.5 times 10 to the power of 6 for BPE, declining significantly with CAR A2 and CAIleR 8 Treg treatment. Panel E shows scatter dot plots of immune cell percentages in BALF, where eosinophils reach approximately 60 percent in BPE and decline significantly with both CAR A2 and CAIleR 8 Treg treatment, while neutrophils and lymphocytes show smaller changes. Panel F shows IL-10 levels in BALF reaching approximately 200 picograms per milliliter for PBS, declining with BPE and partially recovering with CAR A2 and CAIleR 8 Treg treatment. Panel G shows scatter dot plots of neutrophil, monocyte, and eosinophil percentages gated on CD11b positive cells, where eosinophils decline significantly with CAR A2 and CAIleR 8 Treg treatment. Panel H shows SiglecF expression on eosinophils with higher expression declining significantly with CAR A2 and CAIleR 8 Treg treatment. Panel I shows CD4 positive T cell percentages for IL4, IL5, IL13, and IL17, where CAR A2 and CAIleR 8 Treg treatment reduces IL4, IL13 and IL17. Panel J shows hematoxylin and eosin as well as PAS stained lung histology images across PBS, BPE, CAR A2 Treg, and CAIleR 8 Treg conditions, with Panel K showing PAS positive percent declining from approximately 60 percent in BPE to approximately 30 percent with CAIleR 8 Treg treatment with significant differences marked. Panel L shows line graphs of respiratory mechanics parameters Rn, G, and H across methacholine concentrations from baseline to 50 milligrams per milliliter, where CAIleR 8 Treg shows significantly lower G and H values at 50 milligrams per milliliter compared to CAR A2 Treg.
CAlleR 8 murine Tregs for the treatment of allergic airway inflammation. (A) CAR A2 and CAlleR 8 murine constructs. Experimental design for murine CAlleR Treg production. (B) Representative flow cytometry plots and cumulative data of CAlleR Treg phenotype after 7 days of expansion with CD25 and Foxp3 expression to assess purity (n = 4–6) and Thy1.1 and Bet v1 expression for transduction efficiency (n = 4–7). Cumulative data of Treg expansion after 7 days of culture. (C)In vivo experimental design of BPE-induced allergic airway inflammation. (D) Cell counts in BALF (n = 6–9). (E) Differential counts of cells infiltrating the BALF (n = 6–9). (F) IL-10 quantification in BALF by ELISA. (G) Percentages of granulocytes in the lungs (n = 7–9). (H) SiglecF expression on eosinophils in the lungs (n = 4–9). (I) Th2 cytokine production in CD4+ T cells of the lungs (n = 7–10). (J) Representative images of H/E and PAS staining of the lungs (black bars represent 100-μm scale). (K) Quantification of PAS+ small and medium airways of the lungs (n = 7–9). (L) FlexiVent measurement of Newtonian resistance (Rn), tissue elastance (H), and tissue damping (G) at baseline and at different doses of inhaled methacholine (n = 4–5). Data are presented as the mean ± SEM from two independent experiments (except for L that shows data from one experiment). n indicates the number of mice. P values were determined by one-way ANOVA with Tukey’s multiple comparison test in D, E, F, G, I, and K and two-way ANOVA with Tukey’s multiple comparison test in H and Sidak’s test in L. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. H/E, hematoxylin/eosin.