Figure 4. Reduced CD18 function disrupts cell-cell contacts between DCs and Cd18^hypo Tregs from PL/J mice, which impairs specific allogeneic Treg expansion and activation.

(A) Representative pictures of cluster formation of allogeneic DCs with Tregs derived from either Cd18^wt mice or Cd18^hypo mice are shown. Original magnification, ×40. (B) Cluster formation between allogeneic DCs and Tregs of different genotypes from Cd18^wt mice and Cd18^hypo mice was assessed by counting aggregated clusters/HPF in 100 randomly selected HPFs. Cluster formation with allogeneic DCs was substantially reduced for Tregs derived from Cd18^hypo mice compared with Tregs from Cd18^wt control mice. **P = 0.0029, using Student’s t test. (C) Increased neutralizing mAb against CD18 resulted in decreased proliferative response of specific allogeneic Tregs in MLRs. Numbers on the top left of C and F indicate the percentage of CFSE-labeled proliferating cells. Numbers on the top right of C and F indicate the percentage of undivided CFSE-labeled cells. (D) Increased TGF-β1 expression by Cd18^wt Tregs was observed in MLRs. Neutralizing mAb against CD18 in MLRs resulted in a dramatic decrease in TGF-β1 expression compared with isotype-matched control antibody. Gray region, TGF-β1 expression; white region, normal goat IgG control for TGF-β1 staining. Numbers on the top of D indicate the percentage of CFSE-labeled proliferating cells. CD4⁺CD25⁺CD127^– Tregs were purified from 4 pooled spleens of Cd18^wt PL/J mice and cocultured with irradiated allogeneic DCs in the presence of 500 units/ml recombinant murine IL-2 and various concentrations of anti-CD18 mAb (E), or anti-mouse CD11a mAb (F), or isotype-matched IgG for 7 days. Tregs were then separated from allogeneic DCs by CD11c MACS beads, extensively washed 3 times with PBS, and mixed at a ratio of 1:4 with Cd18^wt Tresp cells. After 3 days of culture, cells were harvested and analyzed by flow cytometry. One representative experiment out of 3 or 4 independent experiments is shown.