(A) Frequency of cell-to-cell contacts between CD18^Foxp3 or CD18^wt Treg (green) and WT BMDC (blue) assessed by time-lapse microscopy for 2 hours. Individual Treg of 4 different picture sections were selected, and cell contacts with BMDC were counted with ImageJ analysis software. Total number of Treg in each section was related to the number of contacts; n = 2. (B) Interaction time of CD18-deficient Treg with BMDC. Ten different picture sections were counted every 6 minutes up to 60 minutes to determine length of contacts. (C and D) Quantification of the number and average size of DC-Treg aggregates/clusters after 24 hours of culture, measured using ImageJ analysis software. Two experiments were pooled; n = 2. Representative pictures of cluster formation between Treg (green) and DC ( blue) cocultures of CD18^wt and CD18^Foxp3 mice. Magnification, 10×. (E) Representative experiment of cell-to-cell contacts between CD18^Foxp3 or CD18^wt Treg (green) and WT conventional T cells (red) assessed by time-lapse microscopy. Percentage of contacts related to total cell number was determined as described in A; n = 2. (F–H) Flow cytometric quantification of DC expression of CD86, IL-2, and IL-12 in coculture with Treg; n = 3. (I) Secreted IL-1β and IL-6 measured by cytometric bead array in supernatant of DC-Treg cocultures; n = 3. (J–M) Quantification of flow cytometry data showing total frequencies of cDC1 and cDC2 and activation status by proportion of CD86⁺ cells and geometric mean of the fluorescence intensity (gMFI) of CD40 (L) and ICAM (M) in control and CD18^Foxp3 mice; n = 4. Dots represent individual mice. Data are shown as mean ± SD (A–I) or SEM (J–M). Significance was determined by 2-tailed unpaired t test (A–H) or 2-way ANOVA with Šídák’s multiple-comparison test (I–M). *P < 0.05; **P < 0.01; ***P < 0.001; ****P ≤ 0.0001.