Figure 6. Tfr cells regulate Tfh13-mediated IgE responses in vitro.

a) Schematic of experimental design for an in vitro HDM suppression assay. Total B, Tfh and Tfr cells were purified from the dLN of mice that received HDM on days 0,2,4 and 6 and added to culture wells along with HDM for 6 days.

b) Quantification of total Tfh cells (left) and the percentage of Tfr cells (FoxP3⁺ of CD4⁺IA-CD19- cells) (right) from cultures as in (a).

c) Quantification of cytokines in culture supernatants from cultures as in (a). Cytokines listed in red have levels statistically lower in cultures containing Tfr cells compared to cultures without Tfr cells.

d) Column graphs of IL5, IL4 and IL13 from data in (c).

e) Intracellular cytokine staining of IL-13 in Tfh cells from cultures as in (a).

f) Analysis of class switching to IgG1 and IgE in cultures as in (a). Representative gating (left, pregated on CD19⁺IA⁺CD4- cells), IgE⁺ B cell quantification (middle) and IgG1⁺ B cell quantification (right) are shown.

g) Counts of total IgE⁺ (left) and IgG1⁺ (right) B cells expressed as relative counts from experiments as in (a).

h) Expression of GL7 on IgE⁺ B cells from indicated cultures as in (a).

i) Expression of IgE on IgE⁺ B cells (left) and IgG1 on IgG1⁺ B cells are shown from cultures as in (a).

j) Levels of IgE (left) and IgG (right) in culture supernatants from cultures as in (a). Anti-IL13 or anti-IL4 were added to indicated wells.

Column graphs represent the mean with error bars indicating standard error. P value indicates two-tailed student’s T test (b-i, j; right) or One-way ANOVA with Tukey’s correction (j; left).

Data are from individual experiments and are representative of 3 independent experiments.