Figure 1. LAG-3 blockade enhances the accumulation of HA-specific CD8⁺ cells in a model of self tolerance.

We transferred 10⁶ LAG-3^+/+ or LAG-3^–/–CD8⁺Thy1.1⁺ clone 4 CD8⁺ cells into Thy1.2⁺C3-HA^high mice. Mice receiving antibody were given 0.2 mg αLAG-3 i.p. at the time of transfer and 3 days later. At indicated time points after transfer, single-cell suspensions were made from lungs and analyzed for LAG-3 expression and function. (A) CD8⁺ and Thy1.1⁺ were gated to determine expression of LAG-3 on CD8⁺ T cells taken directly from lungs of C3-HA mice. Both surface expression (left) and intracellular staining (middle and right) were performed for LAG-3 expression. Endogenous (endo) CD8 populations within lungs were also assessed. Blue lines, LAG-3; red lines, rat IgG1 isotype; black lines, LAG-3^–/– cells. Max, maximum. (B) Lungs from C3-HA mice were analyzed 7 days after transfer for percentage of CD8⁺Thy1.1⁺ cells by staining for Thy1.1 (C). Clonotypic cells from B were analyzed for division by dilution of CFSE and IFN-γ production by intracellular staining after stimulation in vitro in the presence of HA peptide plus monensin for 5 hours. SSC-H, side scatter. (D and E) Absolute numbers of clonotypic and IFN-γ⁺ clonotypic cells/lung after αLAG-3 treatment or transfer of LAG-3^–/– clone 4 cells. The mean from 3 mice per group is shown. Each experiment was performed 3 times with data from 1 representative experiment shown.