Panel A shows sequential gating flow cytometry plots progressing through cells at 60.2 percent, single cells at 75.2 percent, alive at 40.9 percent, CD45 positive at 92.7 percent, and CD11b positive at 55.6 percent, followed by neutrophil gating at 26.3 percent and Ly6G negative at 73.7 percent, monocyte gating at 8.59 percent and Ly6C negative cells at 84.5 percent, followed by eosinophil gating at 93.2 percent. Gating for eosinophil SiglecF high and low is shown for all conditions. Panel B shows absorbance at 450 nanometers for Bet v1 IgG1 where BPE reaches approximately 1.6 and CAR A2 and CAIleR 8 Treg declines to approximately 0.8 with significant differences. Panel C shows IgE levels in micrograms per milliliter remaining near 0.5 to 1 across conditions with significant difference marked between PBS and BPE. Panel D shows flow cytometry gating for dendritic cells through CD11c, F4/80, and MHC-II axes, with a scatter dot plot showing DC percent in lung remaining around 40 to 60 percent across conditions where mice were sensitized. Panels E and F show scatter dot plots of T cell population percentages across CD3, CD4, CD8, Foxp3, and CD44 markers in lung and mediastinal lymph node, where CD44 expression was reduced in CD4 positive cells in CAR A2 and CAlleR 8 Treg conditions. Histograms showing CD44 rightward shifts in BPE compared to PBS. Panel G shows histograms of IL4, IL5, IL13, and IL17 counts across PBS, BPE, CAR A2, and CAIleR 8 conditions, where BPE shows elevated cytokine positive percentages of approximately 6 to 9 percent for IL4, IL5, IL13, and IL17, declining with CAR A2 and CAIleR 8 treatment to approximately 4 to 6 percent.
CAlleR 8 Tregs do not regulate immunoglobulin production or affect the T cell and dendritic cell compartments in the treatment model. (A) Representative flow cytometry plots showing the gating strategy for neutrophils, monocytes, and eosinophils in the lungs. The expression of SiglecF in lung eosinophils is also shown. (B) Specific anti-Bet v1 IgG1 detection in mouse serum by ELISA (n = 7–9 from two independent experiments). (C) Total serum IgE quantification by ELISA (n = 6–10 from two independent experiments). (D) Gating strategy and cumulative data on the percentage of DCs detected in the lungs (gated on CD11c+CD11b+F480−MHC-II+) (n = 7–10 from two independent experiments). (E and F) Cumulative data of the percentage of the different T populations observed in (E) the lungs and in (F) the mLN (CD3+ T cells, CD4+, CD8+, and Tregs) and representative plots and cumulative percentage of CD4+ T cells expressing the activation marker CD44 (n = 7–10 from two independent experiments). (G) Representative flow cytometry plots showing intracellular expression of Th2 cytokines in lung CD4+ T cells. Data are represented as the mean ± SEM, and P values were calculated with one-way ANOVA with Tukey’s multiple comparison test in B, C, and D and with two-way ANOVA with Tukey’s multiple comparison test in E and F. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. DCs, dendritic cells.