(A) TLR2 and TCR triggering cooperate to induce Treg expansion in vivo. TLR2^–/– mice were reconstituted with 2 × 10⁶ freshly isolated and CFSE-labeled OT-II–transgenic Tregs (TCR of OT-II transgenic T cells is Vα2 and specific for the OVA-peptide presented in I-A^b). The reconstituted mice were subsequently challenged i.p. with either PAM (20 μg/mouse) or OVA-peptide [OVA-pep] (10 μg/mouse) alone or with the combination of PAM and OVA-peptide. After 4 days, splenocytes were isolated and analyzed by flow cytometry for CFSE-fluorescent signal of the infused cells. The cells shown are gated for the CD4⁺, Vα2⁺, CFSE⁺ cells, and propidium iodide–positive (death) cells were excluded from the analysis. The value indicates the percentage of cells within the proliferative fraction (>1 division). (B and C) TLR2 triggering abrogates Treg-mediated suppression of anti–C. albicans immunity in vivo. TLR2^–/– mice (5 per group) were reconstituted with 4 × 10⁶ WT PAM–expanded Tregs (B) or conventional Th cells (C) and challenged i.v. with 10⁵ live C. albicans cells 1 day later (day 0). If indicated, mice received an i.p. injection of 100 μl saline (controls) or 20 μg PAM/100 μl saline on days –1, 1, 3, and 5. SEVEN days after the challenge, C. albicans outgrowth (CFU/g tissue ± SEM) from kidneys was monitored. (D) Ex vivo IFN-γ production (± SEM) by C. albicans–stimulated splenocytes was measured as described in Methods. Representative results of 2 independent experiments are shown. *P < 0.05 with TLR2^–/– control.