LFA-1 blockade influences cytokine production and increases the ratio of Tregs to Teffs. B6 recipients of BALB/c SG were treated with anti–LFA-1 alone, CTLA-4 Ig alone, or a combination of anti–LFA-1 plus CTLA-4 Ig, and graft-draining LNs were isolated 9 days after skin grafting. Cells were restimulated with irradiated BALB/c responders for 4 hours and stained intracellularly for cytokines as described in “Methods.” (A) Data shown are gated on CD8⁺ T cells and are representative of 3 independent experiments with 8-9 mice per group. (B) Absolute numbers of double-positive (TNF⁺IFNγ⁺), and single IFNγ⁺ producers in draining LNs are shown. Data shown are combined from 2 independent experiments with a total of 3-6 mice per group. A statistically significant decrease in both IFN-γ⁺TNF⁺ double producers and IFN-γ⁺ single producers was observed only in animals that received both anti–LFA-1 and CTLA-4 Ig, (P < .0001 compared with untreated and P < .05 compared with anti–LFA-1 alone). L indicates anti–LFA-1; C, CTLA-4 Ig. (C) The ratio of CD4⁺CD25⁺FoxP3⁺ T cells to IFN-γ-secreting Teffs was statistically significantly increased in LFA-1–treated animals (P = .0095) and in LFA-1 + CTLA-4 Ig–treated animals (P = .0043) compared with untreated controls. Data shown are summarized from 2 experiments with a total of 3-6 mice per group.